Intracoronary Device And Method Of Use Thereof

ABSTRACT

Engraftment of therapeutic cells and agents to a target site in an organism is enhanced by mechanical, chemical and biological methods and systems.

TECHNICAL FIELD

This document relates generally to delivery of therapeutic cells to atarget site of a mammal and in particular, to a method and apparatus forenhancing engraftment at the target site.

BACKGROUND OF THE INVENTION

Damaged tissue, such as a lesion in a vessel, can be treated withtherapeutic cells. For example, therapeutic cells can be injected intothe vasculature to treat a lesion in the vessel. Some therapeutic cellswill attach to the target site and provide treatment to the damagedtissue. However, depending on factors such as the dimensions of thetarget site, some of the therapeutic cells will flow past the lesionsite without attaching to the site. Those therapeutic cells that fail toattach provide no benefit. Moreover, it has been reported thatautologous bone marrow cells isolated from patients with chronic heartfailure have “significantly reduced migratory and colony formingactivity in vitro and a reduced neovascularization capacity in vivo”compared to cells from healthy controls (Circulation, 2004; 109:1615-1622). The inability of such cells to migrate may lead to limitedengraftment and colony forming activity may contribute to “limitedtherapeutic potential.”

What is needed are methods and systems for improving the engraftment oftherapeutic cells at a target site or region, e.g., a region of damagedtissue.

SUMMARY OF THE INVENTION

Various embodiments of the present subject matter provide enhancedmigratory function, enhanced adhesion probability, increased residencetime (for example longer residence time of the therapeutic cell in thecoronary arteries), increased engraftment and increased likelihood oftherapeutic potential. The methods and system disclosed herein includeadding agents or secondary processing aimed at improving delivery andengraftment of delivered cells.

The efficiency of cell delivery and engraftment depends on factorsincluding the infusion regimen, local milieu and the state of the cell.

For example, the infusion regimen includes such considerations as theshear rate and the cell residency time. In addition, the local milieuincludes considerations such as the homing gradient, the presence ofendothelial cells adhesion molecules, presence of bone marrow adhesionmolecules and improved vessel permeability. Furthermore, the state ofthe cell is a function of cell viability, concentration and presence ofadhesion molecules.

The invention comprises a method of enhancing engraftment of therapeuticcells at a target site in a mammal comprising conditioning the cells toprovide cells having an altered number of adhesion molecules as comparedto corresponding cells not subjected to the conditioning, wherein theconditioning increases the probability of engraftment of the cell at thetarget site; and delivering a composition comprising the conditionedtherapeutic cells to the target site using a intercoronary deliverydevice.

The therapeutic cells of the present invention may comprise pluripotent,totipotent cells, autologous cells, non-autologous cells, or xenogeniccells. The methods of the present invention comprise conditioning thecells via biological conditioning, chemical conditioning, mechanicalconditioning, or any combination thereof.

The methods of the invention comprise biological conditioning whichcomprises contacting the cell with at least one chemokine, cytokine,growth factor, or exogenous agent. The methods of the inventioncomprising biological conditioning which comprises subjecting the cellsto periods of hypoxia. The methods of the present invention furthercomprise conditioning cells associated with the target site to providetarget cells having an altered number of adhesion molecules as comparedto corresponding target cells not subjected to the conditioning, whereinthe conditioning increases the probability of engraftment of thetherapeutic cells at the target site.

The present invention provides a method of enhancing engraftment of atherapeutic cell at a target site in a mammal comprising delivering acomposition comprising the therapeutic cell and one or more engraftmentenhancing agents, wherein the composition is delivered to the targetsite using an intercoronary delivery device.

The present invention provides a method of enhancing engraftment of atherapeutic cell at a target site in a mammal comprising delivering acomposition comprising the therapeutic cell and one or more engraftmentenhancing agents, wherein the composition is delivered to the targetsite using an implantable delivery device, and wherein the one or moreengraftment enhancing agents is biocompatible and provides transient,localized ischemia at the target site.

The present invention further provides a method of enhancing engraftmentof a therapeutic cell at a target site in a mammal comprising deliveringa composition comprising the therapeutic cell and one or moreengraftment enhancing agents, wherein the composition is delivered tothe target site using an implantable delivery device, and wherein theone or more engraftment enhancing agents is biodegradable and providestransient, localized ischemia at the target site.

The present invention also provides a method of enhancing engraftment ofa therapeutic cell at a target site in a mammal comprising subjectingthe therapeutic cell to in vitro conditioning, wherein the conditioningincreases the probability of engraftment of the therapeutic cell at thetarget site; and delivering a composition comprising the conditionedtherapeutic cell, wherein the composition is delivered to the targetsite using an implantable delivery device.

The present invention provides a catheter comprising a catheter bodyhaving a dual lumen, a mixing chamber at a terminus of the catheterbody, the mixing chamber having an outlet, a porous material coupled toa first lumen to generate bubbles within the mixing chamber, a dischargeport coupled to the second lumen to introduce a cell into the mixingchamber, and a bypass port to admit blood into the mixing chamber.

The present invention also provides a method comprising inducingischemia at a target site for a transitory period of time, delivering atherapeutic cell and a viscous agent to the target site, the viscousagent selected to increase a viscosity of the therapeutic cell, andrestoring normal blood flow to the target site.

The present invention provides a method of delivering a therapeutic cellto a target site in a mammal comprising introducing a solution includingthe therapeutic cell and an agent, wherein the agent is tailored toenhance engraftment of the therapeutic cell to the target site, andwherein the solution is introduced using an implantable catheter.

Also provided by the present invention is a method comprising modifyinga target cell to upregulate an adhesion molecule counter-receptor,subjecting a therapeutic cell to mechanical conditioning so as toprovide an increased number of adhesion molecules on the cell surface ascompared to a non-conditioned cell, and delivering the therapeutic cellto the site of the target cell.

The present invention provides a method comprising combining a magneticparticle and a therapeutic cell, applying a static magnetic field to atarget site of a mammal, the static magnetic field having a gradientoriented in a direction normal to a vessel wall at the target site, andintroducing the therapeutic cell.

The present invention further provides a method of enhancing engraftmentof therapeutic cells at a target site in a mammal comprising contactingthe therapeutic cells with a biological linker, wherein at the linker isattached to the cell membrane of a therapeutic cell, and wherein atleast one functionality of the linker molecule has affinity to thesurface of the therapeutic cell, and wherein at least one otherfunctionality of the linker has affinity to the surface of the lumensurface of the target area vasculature.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, like numerals describe similar components throughoutthe several views. Like numerals having different letter suffixesrepresent different instances of the components.

FIGS. 1A, 1B and 1C illustrate views of a shear module.

FIG. 1D illustrates surface modified therapeutic cells.

FIG. 1E illustrates a therapeutic cell attached to a lumen surface by abi-functional linker molecule.

FIG. 2A illustrates surface modified therapeutic cells.

FIG. 2B illustrates a therapeutic cell attached to a lumen surface by anN-hydroxy succinimide (NHS) reactive linker molecules.

FIG. 3 illustrates an externally applied magnetic field gradient and anorgan.

FIG. 4A illustrates surface modified endothelial cells.

FIG. 4B illustrates therapeutic cells attached to a lumen surface bybi-functional linker molecules.

FIG. 5A illustrates endothelial cells attached to NHS reactive linkermolecules.

FIG. 5B illustrates therapeutic cells attached to a lumen surface by NHSreactive linker molecules.

FIG. 6A illustrates a system having a dual lumen catheter body and abubble producing mixing chamber.

FIG. 6B illustrates a bubble producing mixing chamber.

DETAILED DESCRIPTION

Reference will now be made in detail to certain claims of the invention,examples of which are illustrated in the accompanying text and examples.While the invention will be described in conjunction with the enumeratedclaims, it will be understood that they are not intended to limit theinvention to those claims. On the contrary, the invention is intended tocover all alternatives, modifications, and equivalents, which may beincluded within the scope of the present invention as defined by theclaims.

I. Definitions

An “engraftment enhancing agent” is defined herein as an agent orprocess of cellular manipulation that promotes, improves or enhancescellular engraftment of a therapeutic cell at a target site, forexample, an agent that enhances the incorporation, i.e., adherenceand/or transmigration, of a therapeutic cell in an area of infarctedmyocardium. A process of cellular manipulation that enhances theincorporation of a therapeutic cell in a target site includes, forexample, subjecting the therapeutic cell to conditioning, e.g.,mechanical conditioning such as shear.

A “therapeutic cell” is appropriate cellular material introduced intoand/or in the vicinity of damaged tissue. For example, a “therapeuticcell” includes, but is not limited to, a pluripotent or totipotent cell,e.g., a cell having broad developmental potential and “plasticity,” forexample, an “adult” stem cell, i.e., a post-natal stem cell, forexample, a multipotent adult progenitor cell, or a cell derived from thebone marrow such as a hematopoietic stem cell (HSC), a hematopoieticprogenitor cell, a non-hematopoietic mesenchymal stem cell (MSC), or astromal cell; an embryonic stem cell, a cell from cord blood, anisolated CD34⁺ cell, fetal cardiomyocytes, skeletal myoblasts,endothelial progenitor cells. By “therapeutic cell” is also meantskeletal muscle derived cells, for instance, skeletal muscle cells andskeletal myoblasts; cardiac derived cells, myocytes, e.g., ventricularmyocytes, atrial myocytes, SA nodal myocytes, AV nodal myocytes, smoothmuscle cells and fibroblasts. In one embodiment, the therapeutic cellsare recombinant cells, such as recombinant CD34⁺ cells. In anotherembodiment, the therapeutic cells are capillary endothelium. In yetanother embodiment, the therapeutic cells are autologous cells includingxenologous cells, however, non-autologous cells may be employed.

By “target cell” is meant a cell located at or in the vicinity of a“target site” in a subject to which a therapeutic cell is directed. A“target site” can be an area or region of vascular damage, disease orinjury, or an area proximal to a region of vascular damage, disease, orinjury. For example, a “target cell” can be an endothelial cell presenton the lumen wall of a patient having myocardial injury or damage, suchas in a patient having experienced myocardial infarction. A “targetsite” also includes vasculature, such as arterial and/or venous otherthan cardiac and/or coronary. In one example, a “target cell” is anendothelial cell residing in the vasculature of the target site.

By “myocardium” is meant the muscular portion of the heart. Themyocardium includes three major types of muscle fibers: atrial musclefibers, ventricular muscle fibers, and specialized excitatory andconductive muscle fibers.

“Ischemia” is a condition where oxygen demand of the tissue is not metdue to localized reduction in blood flow caused by narrowing orocclusion of one or more vessels. “Occlusion” is the total or partialobstruction of blood flow through a vessel. By “transient, localizedischemia” is meant a temporary state of ischemia in a confined area oftissue caused by temporary total or partial obstruction of blood flowthrough a vessel. For example, “transient, localized ischemia” refers toa temporary decrease in blood flow below that needed to maintainadequate tissue oxygenation, also known as a supply demand imbalance ora demand that exceeds supply.

By “homing” or “homing process” is meant the migration of cells, e.g.,therapeutic cells such as stem cells, and attachment to a target site,i.e., a site of injury or ischemia. Once attached, an environment isprovided that is favorable to the growth and differentiation ofcardiomyocytes because of increased vascular permeability, cytokinerelease, and adhesion protein expression. The expression of adhesionmolecules, such as vascular endothelial growth factor (VEGF) and stromalcell-derived factor-1 (SDF-1), is up-regulated in hypoxic tissue.

As used herein, the phrase “adhesion molecules” refers to, for example,ligands and receptors that play a role in inter-cellular adhesion, suchas the initiation of contact (tethering) between a therapeutic cell,e.g., a stem cell, and a target cell, e.g., an endothelial cell. Forexample, an endothelial cell present at the site of vascular injuryexpresses a receptor for a ligand expressed on a stem cell. Exemplary“adhesion molecules” that may be present on a therapeutic cell include,but are not limited to, CD44, P-selectin glycoprotein ligand-1 (PSGL-1;CD 162), hematopoietic cell E-/L-selectin ligand (HCELL), E-selectinligand-1, Very Late Antigen-4 (VLA-4; CD49d), Leukocyte FunctionAssociated Antigen-1 (LFA-1), an integrin, such as an α4 integrin or aβ2 integrin, CD31, VE-Cadherin (CD144), PECAM (CD31), vascular celladhesion molecule-1 (VCAM-1), intercellular adhesion molecule (ICAM)-1,a selectin such as P-Selectin (CD62P), E-Selectin (CD62E), L-selectin,α4β7, Mac-1, and cutaneous lymphocyte antigen. While not involveddirected in inter-cellular adhesion per se, the phrase “adhesionmolecules” also includes receptors that are present on eithertherapeutic cells or target cells, e.g., CD34, CD133, VEGF receptor 1(flt-1/flk-2), VEGF receptor 2 (flk-1/KDR), and CXCR4, that can beutilized to manipulate the adhesion of a therapeutic cell to a targetcell. For example, as discussed herein, bi-functional antibodies thatspecifically bind to CD133 may be utilized to modify the surface of atarget cell.

To migrate to tissue(s), a therapeutic cell must first adhere to thetarget site with sufficient strength to overcome the shear forces ofblood flow in a process known as “rolling.” Once tethered, thetherapeutic cell “rolls” via binding to its corresponding endothelialadhesion molecule.

A “vector” or “construct” (sometimes referred to as gene delivery orgene transfer “vehicle”) refers to a macromolecule or complex ofmolecules comprising a polynucleotide to be delivered to a host cell,either in vitro or in vivo. The polynucleotide to be delivered maycomprise a coding sequence of interest for gene therapy. Vectorsinclude, for example, viral vectors (such as adenoviruses,adeno-associated viruses (AAV), lentiviruses, herpesvirus andretroviruses), liposomes and other lipid-containing complexes, and othermacromolecular complexes (such as polycations, e.g., cationic polymers)capable of mediating delivery of a polynucleotide to a host cell.Vectors can also comprise other components or functionalities thatfurther modulate gene delivery and/or gene expression, or that otherwiseprovide beneficial properties to the targeted cells. Such othercomponents include, for example, components that influence binding ortargeting to cells (including components that mediate cell-type ortissue-specific binding); components that influence uptake of the vectornucleic acid by the cell; components that influence localization of thepolynucleotide within the cell after uptake (such as agents mediatingnuclear localization); and components that influence expression of thepolynucleotide. Such components also might include markers, such asdetectable and/or selectable markers that can be used to detect orselect for cells that have taken up and are expressing the nucleic aciddelivered by the vector. Such components can be provided as a naturalfeature of the vector (such as the use of certain viral vectors whichhave components or functionalities mediating binding and uptake), orvectors can be modified to provide such functionalities. A large varietyof such vectors are known in the art and are generally available. When avector is maintained in a host cell, the vector can either be stablyreplicated by the cells during mitosis as an autonomous structure,incorporated within the genome of the host cell, or maintained in thehost cell's nucleus or cytoplasm.

A “recombinant viral vector” refers to a viral vector comprising one ormore heterologous genes or sequences. Since many viral vectors exhibitsize constraints associated with packaging, the heterologous genes orsequences are typically introduced by replacing one or more portions ofthe viral genome. Such viruses may become replication-defective,requiring the deleted function(s) to be provided in trans during viralreplication and encapsidation (by using, e.g., a helper virus or apackaging cell line carrying genes necessary for replication and/orencapsidation). Modified viral vectors in which a polynucleotide to bedelivered is carried on the outside of the viral particle have also beendescribed (see, e.g., Curiel et al., Proc. Natl. Acad. Sci. USA, 88:8850(1991)).

“Gene delivery,” “gene transfer,” and the like as used herein, are termsreferring to the introduction of an exogenous polynucleotide (sometimesreferred to as a “transgene”) into a host cell, irrespective of themethod used for the introduction. Such methods include a variety ofwell-known techniques such as vector-mediated gene transfer (by, e.g.,viral infection/transfection, or various other protein-based,lipid-based or polymer-based gene delivery complexes) as well astechniques facilitating the delivery of “naked” polynucleotides (such aselectroporation, “gene gun” delivery and various other techniques usedfor the introduction of polynucleotides). The introduced polynucleotidemay be stably or transiently maintained in the host cell. Stablemaintenance typically requires that the introduced polynucleotide eithercontains an origin of replication compatible with the host cell orintegrates into a replicon of the host cell such as an extrachromosomalreplicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. Anumber of vectors are known to be capable of mediating transfer of genesto mammalian cells, as is known in the art.

By “transgene” is meant any piece of a nucleic acid molecule (forexample, DNA) which is inserted by artifice into a cell eithertransiently or permanently, and becomes part of the organism ifintegrated into the genome or maintained extrachromosomally. Such atransgene may include a gene that is partly or entirely heterologous(i.e., foreign) to the transgenic organism, or may represent a genehomologous to an endogenous gene of the organism.

By “transgenic cell” is meant a cell containing a transgene. Forexample, a stem cell transformed with a vector containing an expressioncassette can be used to produce a population of cells having alteredphenotypic characteristics.

The term “wild-type” refers to a gene or gene product that has thecharacteristics of that gene or gene product when isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designatedthe “normal” or “wild-type” form of the gene. In contrast, the term“modified” or “mutant” refers to a gene or gene product that displaysmodifications in sequence and or functional properties (i.e., alteredcharacteristics) when compared to the wild-type gene or gene product. Itis noted that naturally-occurring mutants can be isolated; these areidentified by the fact that they have altered characteristics whencompared to the wild-type gene or gene product.

The term “transduction” denotes the delivery of a polynucleotide to arecipient cell either in vivo or in vitro, via a viral vector andpreferably via a replication-defective viral vector, such as via arecombinant AAV.

The term “heterologous” as it relates to nucleic acid sequences such asgene sequences and control sequences, denotes sequences that are notnormally joined together, and/or are not normally associated with aparticular cell. Thus, a “heterologous” region of a nucleic acidconstruct or a vector is a segment of nucleic acid within or attached toanother nucleic acid molecule that is not found in association with theother molecule in nature. For example, a heterologous region of anucleic acid construct could include a coding sequence flanked bysequences not found in association with the coding sequence in nature,i.e., a heterologous promoter. Another example of a heterologous codingsequence is a construct where the coding sequence itself is not found innature (e.g., synthetic sequences having codons different from thenative gene). Similarly, a cell transformed with a construct which isnot normally present in the cell would be considered heterologous.

By “DNA” is meant a polymeric form of deoxyribonucleotides (adenine,guanine, thymine, or cytosine) in double-stranded or single-strandedform found, inter alia, in linear DNA molecules (e.g., restrictionfragments), viruses, plasmids, and chromosomes. In discussing thestructure of particular DNA molecules, sequences may be described hereinaccording to the normal convention of giving only the sequence in the 5′to 3′ direction along the nontranscribed strand of DNA (i.e., the strandhaving the sequence complementary to the mRNA). The term capturesmolecules that include the four bases adenine, guanine, thymine, orcytosine, as well as molecules that include base analogues which areknown in the art.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides)related by the base-pairing rules. For example, the sequence “A-G-T,” iscomplementary to the sequence “T-C-A.” Complementarity may be “partial,”in which only some of the nucleic acids' bases are matched according tothe base pairing rules. There may be “complete” or “total”complementarity between the nucleic acids. The degree of complementaritybetween nucleic acid strands has significant effects on the efficiencyand strength of hybridization between nucleic acid strands. This is ofparticular importance in amplification reactions, as well as detectionmethods that depend upon binding between nucleic acids.

DNA molecules have “5′ ends” and “3′ ends” because mononucleotides arereacted to make oligonucleotides or polynucleotides in a manner suchthat the 5′ phosphate of one mononucleotide pentose ring is attached tothe 3′ oxygen of its neighbor in one direction via a phosphodiesterlinkage. Therefore, an end of an oligonucleotide or polynucleotide isreferred to as the “5′ end” if its 5′ phosphate is not linked to the 3′oxygen of a mononucleotide pentose ring and as the “3′ end” if its 3′oxygen is not linked to a 5′ phosphate of a subsequent mononucleotidepentose ring. As used herein, a nucleic acid sequence, even if internalto a larger oligonucleotide or polynucleotide, also may be said to have5′ and 3′ ends. In either a linear or circular DNA molecule, discreteelements are referred to as being “upstream” or 5′ of the “downstream”or 3′ elements. This terminology reflects the fact that transcriptionproceeds in a 5′ to 3′ fashion along the DNA strand. The promoter andenhancer elements that direct transcription of a linked gene aregenerally located 5′ or upstream of the coding region. However, enhancerelements can exert their effect even when located 3′ of the promoterelement and the coding region. Transcription termination andpolyadenylation signals are located 3′ or downstream of the codingregion.

A “gene,” “polynucleotide,” “coding region,” or “sequence” that“encodes” a particular protein is a nucleic acid molecule that istranscribed and optionally also translated into a gene product, i.e., apolypeptide, in vitro or in vivo when placed under the control ofappropriate regulatory sequences. The coding region may be present ineither a cDNA, genomic DNA, or RNA form. When present in a DNA form, thenucleic acid molecule may be single-stranded (i.e., the sense strand) ordouble-stranded. The boundaries of a coding region are determined by astart codon at the 5′ (amino) terminus and a translation stop codon atthe 3′ (carboxy) terminus. A gene can include, but is not limited to,cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences fromprokaryotic or eukaryotic DNA, and synthetic DNA sequences. Atranscription termination sequence will usually be located 3′ to thegene sequence.

The term “control elements” refers collectively to promoter regions,polyadenylation signals, transcription termination sequences, upstreamregulatory domains, origins of replication, internal ribosome entrysites (“IRES”), enhancers, splice junctions, and the like, whichcollectively provide for the replication, transcription,post-transcriptional processing and translation of a coding sequence ina recipient cell. Not all of these control elements need always bepresent so long as the selected coding sequence is capable of beingreplicated, transcribed and translated in an appropriate host cell.

The term “promoter region” is used herein in its ordinary sense to referto a nucleotide region comprising a DNA regulatory sequence, wherein theregulatory sequence is derived from a gene which is capable of bindingRNA polymerase and initiating transcription of a downstream (3′direction) coding sequence.

By “enhancer element” is meant a nucleic acid sequence that, whenpositioned proximate to a promoter, confers increased transcriptionactivity relative to the transcription activity resulting from thepromoter in the absence of the enhancer domain.

By “cardiac-specific enhancer element” is meant an element, which, whenoperably linked to a promoter, directs gene expression in a cardiac celland does not direct gene expression in all tissues or all cell types.Cardiac-specific enhancers of the present subject matter may benaturally occurring or non-naturally occurring. One skilled in the artwill recognize that the synthesis of non-naturally occurring enhancerscan be performed using standard oligonucleotide synthesis techniques.

By “operably linked” with reference to nucleic acid molecules is meantthat two or more nucleic acid molecules (e.g., a nucleic acid moleculeto be transcribed, a promoter, and an enhancer element) are connected insuch a way as to permit transcription of the nucleic acid molecule.“Operably linked” with reference to peptide and/or polypeptide moleculesis meant that two or more peptide and/or polypeptide molecules areconnected in such a way as to yield a single polypeptide chain, i.e., afusion polypeptide, having at least one property of each peptide and/orpolypeptide component of the fusion. The fusion polypeptide ispreferably chimeric, i.e., composed of heterologous molecules.

“Homology” refers to the percent of identity between two polynucleotidesor two polypeptides. The correspondence between one sequence and toanother can be determined by techniques known in the art. For example,homology can be determined by a direct comparison of the sequenceinformation between two polypeptide molecules by aligning the sequenceinformation and using readily available computer programs.Alternatively, homology can be determined by hybridization ofpolynucleotides under conditions that form stable duplexes betweenhomologous regions, followed by digestion with single strand-specificnuclease(s), and size determination of the digested fragments. Two DNA,or two polypeptide, sequences are “substantially homologous” to eachother when at least about 80%, preferably at least about 90%, and mostpreferably at least about 95% of the nucleotides, or amino acids,respectively match over a defined length of the molecules, as determinedusing the methods above.

By “mammal” is meant any member of the class Mammalia including, withoutlimitation, humans and nonhuman primates such as chimpanzees and otherapes and monkey species; farm animals such as cattle, sheep, pigs, goatsand horses; domestic mammals such as dogs and cats; laboratory animalsincluding rodents such as mice, rats, rabbits and guinea pigs, and thelike.

By “derived from” is meant that a nucleic acid molecule was either madeor designed from a parent nucleic acid molecule, the derivativeretaining substantially the same functional features of the parentnucleic acid molecule, e.g., encoding a gene product with substantiallythe same activity as the gene product encoded by the parent nucleic acidmolecule from which it was made or designed.

By “expression construct” or “expression cassette” is meant a nucleicacid molecule that is capable of directing transcription. An expressionconstruct includes, at the least, a promoter. Additional elements, suchas an enhancer, and/or a transcription termination signal, may also beincluded.

The term “exogenous,” when used in relation to a protein, gene, nucleicacid, or polynucleotide in a cell or organism refers to a protein, gene,nucleic acid, or polynucleotide which has been introduced into the cellor organism by artificial or natural means, or in relation a cell refersto a cell which was isolated and subsequently introduced to other cellsor to an organism by artificial or natural means. An exogenous nucleicacid may be from a different organism or cell, or it may be one or moreadditional copies of a nucleic acid which occurs naturally within theorganism or cell. An exogenous cell may be from a different organism, orit may be from the same organism. By way of a non-limiting example, anexogenous nucleic acid is in a chromosomal location different from thatof natural cells, or is otherwise flanked by a different nucleic acidsequence than that found in nature.

The term “isolated” when used in relation to a nucleic acid, peptide orpolypeptide refers to a nucleic acid sequence, peptide or polypeptidethat is identified and separated from at least one contaminant nucleicacid, polypeptide or other biological component with which it isordinarily associated in its natural source. Isolated nucleic acid,peptide or polypeptide is present in a form or setting that is differentfrom that in which it is found in nature. For example, a given DNAsequence (e.g., a gene) is found on the host cell chromosome inproximity to neighboring genes; RNA sequences, such as a specific mRNAsequence encoding a specific protein, are found in the cell as a mixturewith numerous other mRNAs that encode a multitude of proteins. Theisolated nucleic acid molecule may be present in single-stranded ordouble-stranded form. When an isolated nucleic acid molecule is to beutilized to express a protein, the molecule will contain at a minimumthe sense or coding strand (i.e., the molecule may single-stranded), butmay contain both the sense and anti-sense strands (i.e., the moleculemay be double-stranded).

The term “recombinant DNA molecule” as used herein refers to a DNAmolecule that is comprised of segments of DNA joined together by meansof molecular biological techniques.

The term “recombinant protein” or “recombinant polypeptide” as usedherein refers to a protein molecule that is expressed from a recombinantDNA molecule.

The term “peptide,” “polypeptide” and “protein” are used interchangeablyherein unless otherwise distinguished.

An “antibody” is a protein of the immune system that recognizes antigensand thereby triggers an immune response. By “antibody fragment” is meanta portion or part of an antibody having an antigen-binding domain.

Specific Embodiments of the Invention

In one specific embodiment, the present invention provides a method ofenhancing engraftment of therapeutic cells at a target site in a mammalcomprising conditioning the cells to provide cells having an alterednumber of adhesion molecules as compared to corresponding cells notsubjected to the conditioning, wherein the conditioning increases theprobability of engraftment of the cell at the target site; anddelivering a composition comprising the conditioned therapeutic cells tothe target site using a intracoronary delivery device.

In another specific embodiment of the present invention, the therapeuticcells comprise pluripotent or totipotent cells. In another specificembodiment of the present invention, the therapeutic cells compriseautologous cells, non-autologous cells, or xenogenic cells. In yetanother specific embodiment of the present invention, the mammal is ahuman.

In one specific embodiment of the present invention, the conditioningcomprises biological conditioning, chemical conditioning, mechanicalconditioning, or any combination thereof.

In one specific embodiment of the present invention, the adhesionmolecule is CD44, P-selectin glycoprotein ligand-1 (PSGL-1; CD 162),hematopoietic cell E-/L-selectin ligand (HCELL), E-selectin ligand-1,Very Late Antigen-4 (VLA-4; CD49d), Leukocyte Function AssociatedAntigen-1 (LFA-1), an integrin, such as an α4 integrin or a β2 integrin,CD31, VE-Cadherin (CD144), PECAM (CD31), vascular cell adhesionmolecule-1 (VCAM-1), intercellular adhesion molecule (ICAM)-1, aselectin such as P-Selectin (CD62P), E-Selectin (CD62E), L-selectin,α4β7, Mac-1, cutaneous lymphocyte antigen, CD34, CD133, VEGF receptor 1(flt-1/flk-2), VEGF receptor 2 (flk-1/KDR), or CXCR4.

In one specific embodiment of the present invention, the surface densityof adhesion molecules on the cells is increased as a result ofconditioning.

In one specific embodiment of the present invention, the conditioningcomprises mechanical conditioning. In another specific embodiment of thepresent invention, the mechanical conditioning comprises subjecting thetherapeutic cells to a mechanical shear. In another specific embodimentof the invention, the mechanical shear is induced by a programmablepump. In another specific embodiment, the mechanical shear in the rangeof about 5 dynes/cm² up to about 100 dynes/cm².

In another specific embodiment of the present invention, theconditioning comprises biological conditioning. In one specificembodiment of the present invention, the biological conditioningcomprises contacting the cell with at least one chemokine.

In one specific embodiment of the present invention, the chemokine isIl-1beta, TNF-alpha, IL-4, IL-8, SDF-1, MIP-1, MCP-1/2/3/4 orlymphoactin.

In another specific embodiment of the present invention, the biologicalconditioning comprises contacting the cell with at least one cytokine.In one specific embodiment of the present invention, the cytokine is aplatelet derived cytokine, granulocyte colony-stimulating factor(G-CSF), oxidized LDL, tumor necrosis factor-alpha, interleukin-1, orstem cell factor (SCF).

In one specific embodiment of the present invention, the biologicalconditioning comprises contacting the cell with at least one growthfactor. In another specific embodiment of the present invention, atleast one growth factor is VEGF, FGF, Insulin Growth Factor (IGF), bFGF,Hepatocyte Growth Factor, acidic fibroblast growth factor, fibroblastgrowth factor-4, fibroblast growth factor-5, epidural growth factor, orplatelet-derived growth factor.

In one specific embodiment of the present invention, the biologicalconditioning comprises contacting the cell with PR39, HIF 1 alpha, HIF 2alpha, Insulin Growth Factor (IGF), VEGF, bFGF, Hepatocyte GrowthFactor, eNOS enhancers, P38 inhibitors, statins or S1P agonists.

In another specific embodiment of the present invention, the biologicalconditioning comprises contacting the cell with an exogenous agent. Inone specific embodiment of the present invention, the exogenous agentcomprises a biological conjugate, linker, or an expression cassetteencoding an adhesion molecule gene product.

In a specific embodiment of the present invention, the biologicalconditioning comprises subjecting the cells to periods of hypoxia.

In one specific embodiment of the present invention, the conditioningcomprises chemical conditioning. In one specific embodiment of thepresent invention, the chemical conditioning comprises conjugating amolecule or molecular moiety to the surface of the cell. In anotherspecific embodiment, the chemical conditioning comprises attaching amolecule or molecular moiety to the surface of the cell. In one specificembodiment of the present invention, the chemical conditioning comprisescontacting the therapeutic cells with at least one irritant. In anotherspecific embodiment of the present invention, the chemical conditioningcomprises contacting the therapeutic cells with at least one stimulant.

In one specific embodiment of the present invention, the compositionfurther comprises a viscous agent. In one embodiment of the invention,the viscous agent is tocopherol, a lipid emulsion such as an emulsifiedvegetable oil, a surfactant, a hydrophilic polymer, or any combinationthereof.

In a specific embodiment of the invention, the composition furthercomprises an activated platelet or a platelet-derived microparticle. Inanother specific embodiment of the invention, the composition furthercomprises a calcium ionophore, oleic acid, histamine, DMSO, histamine,bradykinin, serotonin, thrombin, VEGF, a leukotriene or a vasodilator.In a specific embodiment of the invention, the vasodilator is an ACEinhibitor or a nitrate.

In another specific embodiment of the present invention, the compositionfurther comprises at least one agent that increases bumping frequency.In one specific embodiment of the present invention, the agent toincrease the bumping frequency is a microbubble, a liposome, a lipidvesicle, a vesicle with membranes formed from di-block or tri-blockco-polymers, a platelet-derived microparticle, or a microparticle.

In one specific embodiment of the present invention, the conditioningcomprises contacting the cells with a magnetically responsive particle.In another specific embodiment of the invention, further comprisingapplying an external magnetic field gradient to the mammal followingdelivery of the composition. In a specific embodiment of the presentinvention, the magnetic particle is labeled. In one embodiment of theinvention, the magnetic particle is labeled with CD44, P-selectinglycoprotein ligand-1 (PSGL-1; CD 162), hematopoietic cell E-/L-selectinligand (HCELL), E-selectin ligand-1, Very Late Antigen-4 (VLA-4; CD49d),Leukocyte Function Associated Antigen-1 (LFA-1), an integrin, such as anα4 integrin or a (32 integrin, CD31, VE-Cadherin (CD144), PECAM (CD31),vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesionmolecule (ICAM)-1, a selectin such as P-Selectin (CD62P), E-Selectin(CD62E), L-selectin, α4β7, Mac-1, cutaneous lymphocyte antigen, CD34,CD133, VEGF receptor 1 (flt-1/flk-2), VEGF receptor 2 (flk-1/KDR) orCXCR4.

In one specific embodiment of the present invention, the compositionfurther comprises a gaseous agent. In a specific embodiment of thepresent invention, the gaseous agent induces transient, localizedischemia at the target site. In another embodiment of the presentinvention, the gaseous agent is carbon dioxide.

In one specific embodiment of the present invention, the inventionfurther comprises conditioning cells associated with the target site toprovide target cells having an altered number of adhesion molecules ascompared to corresponding target cells not subjected to theconditioning, wherein the conditioning increases the probability ofengraftment of the therapeutic cells at the target site.

In one specific embodiment of the present invention, the compositionfurther comprises a pharmaceutically acceptable carrier. In one specificembodiment of the present invention, the therapeutic cells are deliveredafter the cells are conditioned.

In a specific embodiment, the present invention provides a method ofenhancing engraftment of a therapeutic cell at a target site in a mammalcomprising delivering a composition comprising the therapeutic cell andone or more engraftment enhancing agents, wherein the composition isdelivered to the target site using an intercoronary delivery device. Inone specific embodiment of the present invention, at least oneengraftment enhancing agent is gaseous and provides transient, localizedischemia at the target site. In one specific embodiment of the presentinvention, the gaseous engraftment enhancing agent is carbon dioxide.

In another specific embodiment of the present invention, at least oneengraftment enhancing agent is a viscous agent. In one specificembodiment of the present invention, the viscous agent is tocopherol, alipid emulsion such as an emulsified vegetable oil, a surfactant, ahydrophilic polymer, or any combination thereof.

In one specific embodiment of the present invention, at least oneengraftment enhancing agent is a bumping agent.

In one specific embodiment of the present invention, at least oneengraftment enhancing agent is an activated platelet or aplatelet-derived microparticle. In one specific embodiment of thepresent invention, at least one engraftment enhancing agent is a calciumionophore, oleic acid, histamine, DSMO, a vasodilator or any combinationthereof. In one specific embodiment of the present invention, thevasodilator is an ACE inhibitor or a nitrate.

In one specific embodiment of the present invention, at least oneengraftment enhancing agent is an agent that increases bumpingfrequency. In one specific embodiment of the present invention, theagent to increase the bumping frequency is a microbubble, a liposome, alipid vesicle or a vesicle with membranes formed from di-block ortri-block co-polymers.

In one specific embodiment of the present invention, at least oneengraftment enhancing agent is a chemokine. In another specificembodiment of the present invention, the chemokine is IL-1β, TNF-α,IL-4, IL-8, SDF-1, MIP-1, MCP-1/2/3/4 or lymphoactin. In a specificembodiment of the present invention, the chemokine is SDF-1.

In one specific embodiment of the present invention, at least oneengraftment enhancing agent is a cytokine. In a specific embodiment ofthe present invention, the cytokine is a platelet derived cytokine,granulocyte colony-stimulating factor (G-CSF), oxidized LDL, tumornecrosis factor-alpha, interleukin-1, or stem cell factor (SCF).

In one specific embodiment of the present invention, at least oneengraftment enhancing agent is a growth factor. In yet another specificembodiment of the present invention, at least one growth factor is VEGF,FGF Insulin Growth Factor (IGF), bFGF, Hepatocyte Growth Factor, acidicfibroblast growth factor, fibroblast growth factor-4, fibroblast growthfactor-5, epidermal growth factor, or platelet-derived growth factor.

In one specific embodiment of the present invention, at least oneengraftment enhancing agent is a magnetically responsive particle. Inone specific embodiment of the present invention, the invention furthercomprises modifying the target site to include magnetically responsiveparticles.

In one specific embodiment of the present invention, the inventionfurther comprises applying an external magnetic field gradient to themammal following delivery of the composition. In one specific embodimentof the present invention, the magnetic particle is labeled. In onespecific embodiment of the present invention, the magnetic particle hasa receptor for CD34, CD133, CD44, P-selectin glycoprotein ligand-1(PSGL-1; CD 162), hematopoietic cell E-/L-selectin ligand (HCELL),E-selectin ligand-1, Very Late Antigen-4 (VLA-4; CD49d), LeukocyteFunction Associated Antigen-1 (LFA-1), an integrin, such as an α4integrin or a 132 integrin, CD31, VE-Cadherin (CD144), VEGF receptor 2(KDR), CXCR4, α4β7, Mac-1, or cutaneous lymphocyte antigen.

In another specific embodiment, the invention provides a method ofenhancing engraftment of a therapeutic cell at a target site in a mammalcomprising delivering a composition comprising the therapeutic cell andone or more engraftment enhancing agents, wherein the composition isdelivered to the target site using an implantable delivery device, andwherein the one or more engraftment enhancing agents is biocompatibleand provides transient, localized ischemia at the target site.

In one specific embodiment of the present invention, the biocompatibleengraftment enhancing agent is a liposome.

In one specific embodiment, the present invention provides a method ofenhancing engraftment of a therapeutic cell at a target site in a mammalcomprising delivering a composition comprising the therapeutic cell andone or more engraftment enhancing agents, wherein the composition isdelivered to the target site using an implantable delivery device, andwherein the one or more engraftment enhancing agents is biodegradableand provides transient, localized ischemia at the target site.

In one specific embodiment of the present invention, the biodegradableengraftment enhancing agent includes a microsphere or a microbubble. Inanother embodiment, the microsphere is made of polycaprolactone, PLGApoly(lactide-co-glycolide), polyester-amide, polyphosphazine, ortyrosine carbonate.

In one specific embodiment of the present invention, the microsphere ismade of alginate crosslinked with divalent Ca, Ba or Sr cations.

In one specific embodiment of the present invention, the microspherecomprises an extra-cellular matrix protein crosslinked withglutaraldehyde.

In one specific embodiment, the present invention provides a method ofenhancing engraftment of a therapeutic cell at a target site in a mammalcomprising subjecting the therapeutic cell to in vitro conditioning,wherein the conditioning increases the probability of engraftment of thetherapeutic cell at the target site, and delivering a compositioncomprising the conditioned therapeutic cell, wherein the composition isdelivered to the target site using an implantable delivery device.

In one specific embodiment, the present invention provides a cathetercomprising a catheter body having a dual lumen, a mixing chamber at aterminus of the catheter body, the mixing chamber having an outlet, aporous material coupled to a first lumen to generate bubbles within themixing chamber, a discharge port coupled to the second lumen tointroduce a cell into the mixing chamber, and a bypass port to admitblood into the mixing chamber.

In one specific embodiment of the present invention, the first lumen isconfigured to receive a gas. In another specific embodiment of thepresent invention, also included is a pump configured to generate thebubbles within the mixing chamber at a first predetermined time.

In one specific embodiment of the present invention, the catheterincludes a pump configured to deliver the cell to the mixing chamber ata second predetermined time. In another specific embodiment of thepresent invention, the porous material includes a sponge.

In one specific embodiment, the present invention provides a methodcomprising inducing ischemia at a target site for a transitory period oftime, delivering a therapeutic cell and a viscous agent to the targetsite, the viscous agent selected to increase a viscosity of theinjection medium of the therapeutic cell, and restoring normal bloodflow to the target site.

In one specific embodiment of the present invention, inducing ischemiaincludes introducing a flow resistor. In one specific embodiment of thepresent invention, inducing ischemia includes delivering an irritant orstimulant to the target site.

In one specific embodiment of the present invention, the viscous agentincludes at least one of microspheres, PEG, vitamin E, PVA, PVP,dextran, and dextran sulfate.

In one specific embodiment, the present invention provides a method ofdelivering a therapeutic cell to a target site in a mammal comprisingintroducing a solution including the therapeutic cell and an agent,wherein the agent is tailored to enhance engraftment of the therapeuticcell to the target site, and wherein the solution is introduced using animplantable catheter.

In one specific embodiment of the present invention, the agent inducestransient, localized ischemia at the target site. In one specificembodiment of the present invention, the agent includes at least one ofa microparticle, a liposome and a CO₂ bubble.

In one specific embodiment, the present invention provides a methodcomprising modifying a target cell to upregulate an adhesion moleculecounter-receptor, subjecting a therapeutic cell to mechanicalconditioning so as to provide an increased number of adhesion moleculeson the cell surface as compared to a non-conditioned cell, anddelivering the therapeutic cell to the site of the target cell.

In one specific embodiment of the present invention, the modifyingincludes inducing ischemia.

In one specific embodiment of the present invention, inducing ischemiaincludes introducing a flow resistor in a vessel coupled to the targetsite.

In one specific embodiment of the present invention, shearing includesagitating with a fluid pump.

In one specific embodiment, the present invention provides a methodcomprising combining a magnetic particle and a therapeutic cell,applying a static magnetic field to a target site of a mammal, thestatic magnetic field having a gradient oriented in a direction normalto a vessel wall at the target site, and introducing the therapeuticcell.

In one specific embodiment, the present invention provides a method ofenhancing engraftment of therapeutic cells at a target site in a mammalcomprising contacting the therapeutic cells with a biological linker,wherein at the linker is attached to the cell membrane of a therapeuticcell, and wherein at least one functionality of the linker molecule hasaffinity to the surface of the therapeutic cell, and wherein at leastone other functionality of the linker has affinity to the surface of thelumen surface of the target area vasculature.

In one specific embodiment of the present invention, the linker isirreversibly attached to the therapeutic cell. In one specificembodiment of the present invention, the linker is reversibly attachedto the therapeutic cell.

In one specific embodiment of the present invention, the linker is abiological conjugate.

In one specific embodiment of the present invention, the linker is amultifunctional linker. In one specific embodiment of the presentinvention, the linker is a bi-functional linker.

In one specific embodiment of the present invention, the linker moleculecomprises at least one of an antibody, an antibody fragment, a peptide,or an affibody.

In one specific embodiment of the present invention, the functionalitiesmay be separated by a spacer. In one specific embodiment of the presentinvention, the spacer is a hydrophilic polymer chain. In one specificembodiment of the present invention, the spacer is PEG. In one specificembodiment of the present invention, the spacer has branches or is ofstar form.

In one specific embodiment of the present invention, the linkercomprises linked antibodies, fragments of antibodies (F_(ab) fragments),affibodies, peptides or other molecules with affinity to receptormolecules on the target surface.

II. Therapeutic Cells of the Invention A. Sources of Therapeutic Cellsfor Cell Therapy

Sources for therapeutic cells in cell-based therapies include adult,neonatal and embryonic sources. Adult cells may be derived from variousorgans, such as skeletal muscle derived cells, for instance, skeletalmuscle cells and skeletal myoblasts; cardiac derived cells, myocytes,e.g., ventricular myocytes, atrial myocytes, SA nodal myocytes, AV nodalmyocytes; bone marrow-derived cells or umbilical cord derived cells,e.g., mesenchymal cells and stromal cells; smooth muscle cells;fibroblasts; or pluripotent cells or totipotent cells, e.g., teratomacells, hematopoietic stem cells, for instance, cells from cord blood andisolated CD34⁺ cells, multipotent adult progenitor cells, adult stemcells and embryonic stem cells. For example, progenitor cells (derivedfrom bone marrow or circulating blood) are capable of differentiatinginto myocytes. Progenitor cells can be used to restore cardiac functionin patients with acute or chronic damage to myocardium. For example,intracoronary treatment of acute myocardial infarct patients usingprogenitor cells has provided improved left ventricle ejection fraction.In one embodiment, the therapeutic cells are autologous cells. Inanother embodiment, the therapeutic cells include non-autologous cells,such as allogenic cells. In yet another embodiment, the therapeuticcells include xenogenic cells. The therapeutic cells can be expanded invitro to provide an expanded population of therapeutic cells foradministration to a recipient. In addition, therapeutic cells may betreated in vitro to induce one or more desirable gene products(transgenes) to the cells. For example, cells may be geneticallymodified to express or release chemokines and/or signal messengers whenin situ. For instance, in one example the transgenic therapeutic cellsinclude a transgene that enhances cellular engraftment, cellularproliferation, cellular survival, cellular differentiation and/orcellular function in the recipient. The transgene may be introduced totherapeutic cells by any means including but not limited to liposomes,micelles, polymeric particles, electroporation, naked DNA, plasmid orviral mediated, for instance, via an adenovirus, adeno-associated virus,retrovirus or lentivirus vector.

Sources of therapeutic cells and methods of culturing those cells areknown to the art. See, for example, U.S. Pat. No. 5,130,141 and Jain etal. (Circulation, 103, 1920 (2001)), wherein the isolation and expansionof myoblasts from skeletal leg muscle is discussed (see also Suzuki etal., Circulation, 104, 1-207 (2001), Douz et al., Circulation, 111-210(2000) and Zimmerman et al., Circulation Res., 90, 223 (2002)).Published U.S. application 20020110910 discusses the isolation of andmedia for long term survival of cardiomyocytes. U.S. Pat. No. 5,580,779discusses isolating myocardial cells from human atria and ventricles andinducing the proliferation of those myocardial cells. U.S. Pat. No.5,103,821 discusses isolating and culturing SA node cells. For SA nodecells, the cells may be co-cultured with stem cells or otherundifferentiated cells. U.S. Pat. No. 5,543,318 discusses isolating andculturing human atrial myocytes. U.S. Pat. Nos. 6,090,622 and 6,245,566discusses preparation of embryonic stem cells, while U.S. Pat. No.5,486,359 discusses preparation of mesenchymal cells.

B. Exemplary Methods of Isolating Therapeutic Cells

1. Bone Marrow Derived Cells and Umbilical Cord Derived Cells

Therapeutic cells derived from bone marrow and umbilical cord may beprepared by protocols known in the art, for example, such as thosedisclosed in U.S. Pat. Nos. 5,486,359 and 5,811,094, and in U.S. Patentapplication publication Nos. 20050008624, 20040136967.

2. Therapeutic Myoblasts and Myocytes

a. Cardiac Tissue

Cardiomyocytes may be prepared by a modification of established methods.In particular, primary myocardial cell isolation is done by modifyingestablished protocols by Nag and Chen, Tissue Cell, 13, 515 (1981) andDlugaz et al., J. Cell Biol., 99, 2268 (1984). Briefly, a heart, e.g.,from an organ therapeutic, is dissected and washed in media. Digestionmedia includes modified Jolicks MEM containing 10 mM HEPES, 10 mMpyruvate, 5 mM L-glutamine, 1 mM nicotinamide, 0.4 mM L-ascorbate, 1 mMadenosine, 1 mm D-ribose, 1 mM MgCl₂, 1 mM taurine, 2 mM DL-carnitine,and 2 mM KHCO₃. The hearts are minced in digestion media with 0.5 mg/mlcollagenase (Worthington) and 100 mM CaCl₂. The tissue is treated withsuccessive digestions for 15 minutes at 37° C. The cells from the firstdigestion are discarded and the next six digestion reactions are pooled.Cells are preplated for 1 hour to remove fibroblasts, then plated inPC-1 (Ventrex)/DME-Hams F12 media.

Alternatively, heart muscle is dissected from the left ventricular freewall and quickly cut into pieces of approximately 1 mm³ using an arrayof razor blades. The pieces are incubated for 12 minutes, while shakingat 37° C. in 25 ml of a solution containing 1-2 μM calcium (LC) 120 mMNaCl, 5.4 mM KCl, 5 mM, MgSO₄, 5 mM pyruvate, 20 mM glucose, 20 mMtaurine, 10 mM HEPES, and 5 mM nitrilotriacetic acid, pH 6.96. Themedium is changed several (about 3) times during the twelve minutes. Thepieces are stirred by bubbling with 100% O₂. After removal of the LCmedium by straining with 300 μm gauze, the pieces are incubated at 37°C. for 45 minutes in LC without nitrilotriacetic acid, and 4 U/ml oftype XXIV protease and 30 μM calcium added, followed by two 45 minuteperiods with the protease omitted and 400 IU/ml collagenase added. Themedium is shaken under an atmosphere of 100% O₂. At the end of thesecond and third 45 minute periods, the solution containing thedispersed cells is filtered through a 300 μm gauze and centrifuged at40×g for 1-2 minutes.

Alternatively, primary ventricular myocytes and cardiac fibroblasts areprepared using a Percoll gradient method as described by Iwaki et al.,J. Biol. Chem., 265, 13809 (1990). Cardiac fibroblasts are isolated fromthe upper band of the Percoll gradient, and subsequently plated in highglucose Dulbecco's modified Eagle's medium supplemented with 10% fetalbovine serum. Myocytes are isolated from the lower band of the Percollgradient and subsequently plated in 4:1 Dulbecco's modified Eagle'smedium; 199 medium, 10% horse serum and 5% fetal bovine serum.

After isolation, the cells may be washed in a medium containing calcium,e.g., 30 μM calcium, and resuspended in culturing media. Such culturemedia can comprise DMEM, BSA, ascorbic acid, taurine, carnitine,creatinine, insulin, penicillin G sodium, and an antibiotic, e.g., DMEMwith the addition of 0.2 g BSA, 0.1 mM ascorbic acid, 50 mM taurine, 16mM carnitine, 50 mM creatine, 0.1 μM insulin, 50 U/ml penicillin Gsodium, and 50 mg/ml streptomycin sulfate. Culture media can alsocomprise DMEM without calcium chloride anhydrous and D-calciumpantothenate.

Omega 3 fatty acids have been shown by Kang & Leaf (Circulation, 94,1774 (1996)) to protect against calcium overload and calcium paradox.Therefore, the culture media may also comprise omega 3 fatty acids, suchas, docosaheanoic acid, eicosapentaenoic acid, eicosatetraynoic acid, orpolyunsaturated fatty acid.

Magnesium (Mg⁺) is also known to be protective against calcium overloadand has been shown to be beneficial in failing human myocardium(Schwinger et al., Am. Heart J., 126, 1018 (1993); Schwinger et al., J.Pharmacol. Exp. Ther., 263, 1352 (1992)). Therefore, the culture mediamay comprise varying concentrations of Mg²⁺, e.g., from 0.1 to 16 mM.

In one embodiment, cardiomyocytes are obtained from a tissue sample froma subject, e.g., a vertebrate subject, and successively exposed to afirst solution with decreasing amounts of CaCl₂. The first solutionfurther includes NaCl, HEPES, MgCl₂, KCl, and sugar at a pH ofapproximately 7.4, e.g., 140 mM NaCl, 10 mM HEPES, 1 mM MgCl₂, 5.4 mMKCl, and 10 mM sugar at a pH of approximately 7.4. The tissue may bedisassociated with an enzyme solution and repeatedly resuspended in asecond solution with increasing amounts of CaCl₂. The second solutionmay further include Earle's modified salt, L-glutamine, sodiumbicarbonate, sodium pentothenate, creatine, taurine, ascorbic acid,HEPES, fetal bovine serum, an antibiotic, and a fatty acid, at a pH ofapproximately 7.4, e.g., sodium bicarbonate at 1250 mg/l, creatine at328 mg/500 ml, taurine at 312 mg/500 ml, ascorbic acid at 8.8 mg, HEPESat 2.383 g/500 ml, fetal bovine serum at 10% v/v, an antibiotic at 5%v/v, and a fatty acid at 1 μM at a pH of approximately 7.4.

In yet another embodiment, the second solution can be used to cultivateisolated cells, e.g., cardiomyocytes, including the steps ofresuspending the isolated cells approximately every 24 hours in thesecond solution. In still another embodiment, the second solution can beused as maintenance or culture media for cells, e.g., cardiomyocytes.

In another embodiment, cardiomyocytes are obtained from a tissue samplefrom a subject, e.g., a vertebrate subject, by cutting the tissue intosmaller pieces and incubating the tissue in a first solution. The firstsolution includes calcium, salts, magnesium sulfate, pyruvate, glucose,taurine, HEPES, and nitrilotriacetic acid, e.g., 1-2 μM CaCl₂, 120 mMNaCl, 5.4 mM KCl, 5 mM MgSO₄, 5 mM pyruvate, 20 mM glucose, 20 mMtaurine, 10 mM HEPES, and 5 mM nitrilotriacetic acid, at a pH ofapproximately 6.96. After the addition of an enzyme, e.g., collagenase,to the first solution, the tissue is further incubated in the solutionand later subjected to centrifugation to obtain isolated cells. Aftershaking the tissue at 37° C. for 12 minutes, and bubbling 100% O₂through the solution, the tissue is incubated in a second solutioncomprising 1-2 μM CaCl₂, 30 μM NaCl, 5.4 mM KCl, 5 mM MgSO₄, 5 mMpyruvate, 20 mM glucose, 20 mM taurine, 10 mM HEPES, and 4 U/ml of adigestive enzyme, and subsequently incubated in a third solutioncomprising approximately 1-2 μM, 30 μM NaCl, 5.4 mM KCl, 5 mM MgSO₄, 5mM pyruvate, 20 mM glucose, 20 mM taurine, 10 mM HEPES, and 4 U/ml of adigestive enzyme. Preferably, 400 U/ml of a digestive enzyme, e.g., atype XXIV protease, such as matrix metalloproteinase 2 or 4, and acollagenase, for example, matrix metalloproteinase 1, 3, or 9, is addedto the third solution and the tissue subjected to centrifugation toobtain isolated cells.

Other solutions to enhance the yield and long-term survival rate ofisolated cardiomyocytes include those in published U.S. application20020110910.

b. Neonatal Skeletal Tissue

To harvest cells from neonatal tissue, muscle tissue is harvested from alimb and placed in a culture dish (65 mm diameter) with 8 ml ofcalcium-free PBS. Muscles are removed under sterile conditions. Allharvested tissue is transferred to a 50 ml conical tube containing 12 mlof tissue dissociation solution (TDS) (DMEM with 5% by weight dispaseand 0.5% by weight collagenase IV) and stirred for approximately onehour in order to dissociate the tissue. The tube is then centrifuged at1200×g for approximately 15 minutes. After removal of the supernatant,cells are resuspended in 20 ml of Ham's F12 with 20 mg of collagenasetype IV and incubated at 37° C. for one hour to allow tissuedissociation. The tube is again centrifuged at 1200 g for 15 minutes,after which the supernatant is removed and the cells are resuspended ingrowth media (GM) (400 ml F12, 100 ml FBS and 100 U/ml penicillin G).Within this cell suspension will likely be fibroblasts in addition tomyogenic precursor cells.

c. Adult Skeletal Tissue

Skeletal muscle may also be harvested from adult tissue and cut intostrips. Unlike neonatal tissue, muscle tissue from adult or aged animalsyields more satellite cells if initially preincubated before completetissue dissociation. The increased activation of satellite cells mayresult from the use of NaN₃ in the preincubation media (PI) (90 ml DMand 10 ml 0.05% NaN₃ in 0.9% saline, where DM is 465 ml DMEM, 35 mlhorse serum and 100 U/ml penicillin G).

To preincubate the muscle tissue, the strips are pinned in a SYLGARD™coated culture dish (35 mm diameter), covered with 2.5 ml of PI, andsterilized by exposure to ultraviolet light for approximately 40minutes. The dishes are then maintained at 37° C. in a water-saturatedatmosphere containing 5% CO₂ for 24 to 72 hours, where optimalpre-incubation times may vary for different muscles.

After pre-incubation, each muscle strip is placed into a 50 ml conicaltube with 15 ml TDS solution and incubated in a shaker bath at 37° C.for approximately 3 hours until complete dissociation is observed.Immediately upon complete tissue dissociation, the tubes are centrifugedat 1200 g for 15 minutes. Subsequently, the supernatant is aspirated andcells are reconstituted with 5 ml GM. As with the cells derived fromneonatal tissue, fibroblasts may be included in the cell suspension.

Alternatively, myogenic cells are released from skeletal musclefragments by serial enzyme treatments. A one hour digestion with 600U/ml collagenase (Sigma, St. Louis, Mo., USA), is followed by a 30minute incubation in Hank's balanced salt solution (HBSS) containing0.1% w/v trypsin (Gibco Lab, Grand Island, N.Y., USA). Satellite cellsare placed in 75 cm² culture flasks (Coster, Cambridge, Mass., USA) inproliferation medium, e.g., 199 medium (Gibco Lab.) with 15% fetalbovine serum (Gibco), 1% penicillin (10,000 U/ml) and 1% streptomycin(10,000 U/ml).

In particular, for human myoblasts, these cells are grown fromtherapeutic human muscle and passaged cells are seeded at 2-3,000 cellsper well in a 96 well cluster plate in Ham F12 medium containing 7.5% upto 20% v/v FCS. The medium may contain varying concentrations of LIF.Cell numbers are counted at times up to 12 days. There is a markedstimulation of proliferation of myoblasts by LIF, e.g., at 30 U/ml. FGFand HBGF also stimulate growth of satellite cells (DiMario et al.,Differentiation, 39, 42 (1988)). TGF-α, also stimulates human cells atconcentrations ranging up to 10 ng/ml.

In one embodiment, to expand skeletal muscle cells, skeletal musclecells are cultured with isolated PDGF, TGF-beta, and/or FGF, e.g., at5-10 ng/ml.]

d. Non-Muscle Therapeutic Cells

Methods to isolate and/or culture non-muscle therapeutic cells, andmethods to induce a muscle cell-specific phenotype to those cells, i.e.,differentiation, are known to the art. For instance, mesenchymal stemcells may be obtained by culturing adherent marrow or periosteal cells.

To induce a cardiac cell-specific phenotype, MSCs cells may becocultured with fetal, neonatal or adult cardiac cells optionally in thepresence of fusigens, extracts of mammalian hearts, one or more growthfactors, one or more differentiating agents, or subjected to mechanicalor electrical stimulation.

Bone marrow is a source for therapeutic cells that have the potential todifferentiate into cardiomyocytes, endothelial cells, in the case ofendothelial progenitor cells, and smooth muscle cells (see, for example,Yoon et al., J. Clin. Invest., 115:326-338 (2005)). To obtain bonemarrow cells, a bone marrow puncture is conducted by sternal or iliacpuncture. After skin disinfection of the part for puncture, atherapeutic is subjected to local anesthesia. Particularly, subpeiosteumis thoroughly anesthetized. The inner tube of a bone marrow punctureneedle is pulled out and a 10 ml syringe containing 5000 U of heparin isattached to the needle. Normally 10-20 ml of the bone marrow fluid isquickly taken by suction and the puncture needle is removed, followed bypressure hemostasis for about 10 minutes. The obtained bone marrow fluidis centrifuged at 1000×g to recover bone marrow cells, which are thenwashed with PBS (phosphate buffered saline). After this centrifugationstep is repeated twice, the obtained bone marrow cells are suspended ina cell culture medium such as A-MEM (a-modification of MEM), DMEM(Dulbecco's modified MEM) or IMDM (Isocove's modified Dulbeccos'smedium) each containing 10% FBS (fetal bovine serum) to prepare a bonemarrow cell suspension.

For the isolation of the bone marrow cells having the potential todifferentiate into cardiomyocytes from the obtained bone marrow cellsuspension, any method can be employed, so long as it is effective atremoving other cells existing in the cell suspension such ashematocytes, hematopoietic stem cells, vascular stem cells andfibroblasts. For example, based on the method described in Pittenger etal., Science, 284, 143 (1999), the desired cells can be isolated bysubjecting the cell suspension layered over Percoll having the densityof 1.073 g/ml to centrifugation at 1100×g for 30 minutes, and the cellson the interface are recovered. Furthermore, a bone marrow cell mixturecontaining the cells having the potential to differentiate intocardiomyocytes can be obtained by mixing the above cell suspension withan equal amount of Percoll solution diluted to 9/10 with 10×PBS,followed by centrifugation at 20000×g for 30 minutes, and recovering thefraction having the density of 1.075-1.060. A bone marrow cell mixtureis diluted into single cell using 96-well culture plates to prepare anumber of clones respectively derived from single cells. The cloneshaving the potential to differentiate into cardiomyocyte can be selectedby the observation of spontaneously beating cells generated by thetreatment.

For the isolation of the bone marrow cells having the potential todifferentiate into endothelial cells from the obtained bone marrow cellsuspension, the method described by Asahara et al., Science, 275:964-967 (1997) or Asahara et al., Circulation Research, 85:221-228(1999)) might be employed.

Umbilical blood is another source for therapeutic cells. To preparethose cells, umbilical blood is separated from the cord, followed byaddition of heparin to give a final concentration of 500 U/ml. Afterthoroughly mixing, cells are separated from the umbilical blood bycentrifugation and resuspended in a cell culture medium, such as α-MEM,DMEM or IMDM, each containing 10% FBS. From the cell suspension thusobtained, cells having the potential to differentiate intocardiomyocytes can be separated using, for example, antibodies.

Fibroblasts are also a source for therapeutic cells.

CD34⁺ cells may be obtained from a population of other cells, e.g., fromblood cells when CD34⁺ cells are isolated from blood or cord blood, bycell labeling with magnetic antibodies and subsequent cell separation ina magnetic field. For example, cells may be separated by using acommercially available cell selection system, such as CliniMACS®(Miltenyi Biotec GmbH).

C. Conditioning Therapeutic Cells to Enhance Engraftment at Target Site

As disclosed herein, the surface of therapeutic cells, e.g., stem cells,may be modified to increase the probability or strength of attachment tothe lumen surface of the target area (e.g., endothelium). A variety ofexogenous stimuli (“conditioning”) may be employed in the methods toenhance engraftment, increase the probability or strength of attachmentof the therapeutic cells to the target site. For instance, therapeuticcells may be treated in vitro or in vivo by subjecting them tomechanical conditioning, biological conditioning, chemical conditioning,or any combination thereof. The conditioning may include continuous orintermittent exposure to the exogenous stimuli. Exogenous agents includethose that enhance the attachment, engraftment, survival,differentiation, proliferation and/or function of therapeutic cells,e.g., stem cells, after transplant to the luminal surface of the targetarea, e.g., endothelium. For example, the surface of a therapeutic cellcan be modified in such a way that the surface density of availableadhesion molecules is altered, e.g., increased, wherein the adhesionmolecule possesses an affinity to the luminal surface of the target areavasculature, e.g., to a corresponding adhesion molecule present on orassociated with the cell surface of an endothelial cell or molecularmoieties thereon. In one example, adhesion molecules have an affinity tothe luminal surface of the target vasculature and are antibodies toreceptor molecules present on the surface of the endothelial cells (forexample, anti-CD31 or anti-ICAM). Examples of such conditioning aredisclosed herein. In another example, platelet factor 4 (PF4) is used toupregulate the expression of CD(49d) and CXCR4 on therapeutic cells,which affects cell adhesion and enhance engraftment at a target site. Luet al., Zhonghua Xue Ye Xue Za Zhi, 24: 467-469 (2003).

1. Mechanical Conditioning

As disclosed herein, therapeutic cells may be influenced in a mannersimilar to other cell types such as endothelial cells and lymphocytes toimprove cell engraftment levels.

As used herein, the phrase “mechanical conditioning” refers to amechanical process that results in the alteration of surface density,gene expression, protein synthesis, and/or the activity of one or moreadhesion molecules on the therapeutic cells. For example, exposure ofendothelial progenitor cells to shear stress increases the expression ofvascular endothelial cadherin. Yamamoto et al., J. Appl. Physiol., 95:2081-2088 (2003). See also Peled et al., J. Clin. Invest., 104:1199-1211 (1999) and Rood et al., Exp. Hematol., 27: 1306-1314 (1999).Thus, in one embodiment, the mechanical conditioning of the therapeuticcells, e.g., stem cells, results in the expression and/or upregulationof the expression of adhesion molecules that facilitate the retention ofthe therapeutic cells to the vascular endothelium.

In one embodiment, the mechanical conditioning of therapeutic cellsresults in the enhanced engraftment of the therapeutic cells at thetarget site. For example, therapeutic cells, such as stem cells, may beexposed to controlled shear rates through a shear module consisting ofthe pre-determined length of tubing connected to a controlled flow ratepump. Exposing therapeutic cells to any positive shear stress, forexample, in the range of about 5 dynes/cm² up to about 100 dynes/cm²,may be useful to upregulate expression of an adhesion molecule. Inaddition to shearing cells before delivery, saline fluid can be used tomechanically condition the therapeutic cells.

Views of an exemplary shear module are illustrated FIGS. 1A, 1B and 1C.For example, FIG. 1A illustrates shear module 100 having pump 110, flowcartridge 130 and collection syringe 140. Pump 110 is illustrated as asyringe pump, however other types of pumps are also contemplatedincluding, for example, vane pumps, diaphragm pumps and gear pumps. Pump110 delivers cell suspension 120 to flow cartridge 130 in a controlledmanner. Discharge from flow cartridge 130 is received by collectionsyringe 140. Syringe 140 can be coupled, for example, to stem celldelivery apparatus (not shown). Collection syringe 140, for example, canbe a syringe having a volume of between 1 and 10 cc.

FIG. 1B illustrates a cut-away view of flow cartridge 130. Flowcartridge 130 receives cell suspension 120 at input port 131 anddischarges cell suspension at output port 133. A plurality of channels,each marked 132, serve to control fluid flow through flow cartridge 130.FIG. 1C illustrates a cross-sectional view of flow cartridge 130 havinga plurality of circular channels 132 disposed about the interior. Cellsuspension 120 entering at port 131 is sheared by the flow processthrough channels 132. Each of channel 132, in one example, includes aplastic tube having a diameter of between 100 and 500 microns. Anexemplary material for channel 132 includes polystyrene. Dimensions andmaterials other than those described herein are also contemplated.

Expression profiles of adhesion molecules can also be manipulated by themagnitude and type of shear stress, i.e., laminar v. turbulent, and timeof exposure to the shear stress. Any mechanism that induces shear stressmight be utilized in the mechanical conditioning of the therapeuticcells.

In one example, the present subject matter provides methods and systemsfor modifying the cells ex vivo (prior to delivery at the site), such asin a catheter laboratory to improve cell engraftment.

2. Biological Conditioning

In addition to mechanical conditioning, therapeutic cells can besubjected to biological conditioning to enhance the engraftment at atarget site. For example, brief periods of incubation (e.g., 4-6 hours)of therapeutic cells with chemokines such as Il-1beta, TNF-alpha andIL-4 induces upregulation of E-selectin, ICAM-1 and VCAM-1 onendothelial cells (Konstantopoulos et al., Advanced Drug DeliveryReviews, 33:141-164 (1998)). Cells can be contacted with chemokines fora determined period of time (e.g., about 1 hour to about 24 hours),washed to remove the residual chemokines, and then infused into thepatient. Other biological conditioning agents include, but are notlimited to, PR39, HIF 1 alpha, HIF 2 alpha, Insulin Growth Factor (IGF),VEGF, bFGF, Hepatocyte Growth Factor, eNOS enhancers, P38 inhibitors,statins and S1P agonists.

In addition, biological conditioning includes subjecting therapeuticcells to exogenous agents, such as biological conjugates, linkers, aswell as to expression cassettes (transgenes) encoding a gene productincluding, but not limited to, an adhesion molecule.

In another embodiment, therapeutic cells are subjected to periods ofhypoxia to upregulate adhesion molecules. For example, cells may beincubated in a portable hypoxic chamber for periods of time, forexample, 30 minutes to 24 hours, before delivery into the patient.

a. Biological Conjugates and Linkers The modification of proteins bylabeling with reporter molecules is known in the art. Therapeutic cellscan be contacted with biological linkers, such as biologically activeentities irreversibly attached to the therapeutic cell (see for example,Krantz, Blood Cells, Molecules and Diseases, 23:58-68 (1997) and/orbiological conjugates, such as bifunctional antibody constructs.Alternatively, biological linkers may be attached through a reversiblebond where the time scale of dissociation is sufficiently long tomediate adhesion between therapeutic and target cells.

In one embodiment, the biological conjugate includes but is not limitedto a bi- or multifunctional linker. For example, bi- or multifunctionallinker molecules may be attached to the cell membrane of a therapeuticcell, where at least one functionality of the linker molecule hasaffinity to the surface of the therapeutic cell, and at least one otherfunctionality has affinity to the surface of the lumen surface of thetarget area vasculature, e.g., endothelial cell surface, as illustratedin FIGS. 1D and 1E. FIG. 1D illustrates surface modification oftherapeutic cells using bifunctional linker molecules. In the figure,the linker molecule includes anti-E and anti-T. FIG. 1E illustrates atherapeutic cell attached to an endothelial cell of the targetvasculature. To enhance accessibility, the functionalities may beseparated by a spacer, such as a hydrophilic polymer chain, e.g., PEG.For multifunctional linkers, the spacer may have branches or be of starform. For example, the surface of the therapeutic cell may be modifiedby a molecule consisting of two linked antibodies, where one antibodyhas affinity to a surface receptor (such as an adhesion molecule) on thetherapeutic cell and where the other antibody has affinity to anendothelial surface receptor (such as an adhesion molecule). Instead ofantibodies, fragments of antibodies (F_(ab) fragments), affibodies (alibrary of proteins with a variable region with recognition capabilitiessimilar to antibodies; as disclosed in U.S. Pat. Nos. 5,831,012,6,534,628 and 6,740,734), peptides or other molecules with affinity toreceptor molecules on the respective target surface may be used.

In one embodiment of the invention, the expression of therapeutic cells'adhesion molecules such as receptors CD34, CD 133 and/or KDR, e.g., stemcells, may be altered, e.g., increased, in order to manipulate theadhesion of the therapeutic cells to target cells. For example, aCD133-antibody linked via a PEG spacer to a CD31-antibody may be used tomodify the surface of a therapeutic cell, e.g., a stem cell. In thiscase, the CD133 antibody has affinity to the CD133 receptor present atthe surface of the stem cell, while the CD31 antibody has affinity tothe endothelial cell surface present on the lumen wall of the targetvasculature. To modify the surface of the stem cell, the cells areincubated with a bifunctional anti-CD133-PEG-anti-CD31 molecule. As theanti-CD133 moiety attaches to the CD133 receptor, the stem cell surfacewill effectively present anti-CD31 antibodies with affinity to thesurface of the endothelial cell found in the target vasculature.Examples of other receptor targets present on endothelial cells ofmicrovasculature include, but are not limited to, PECAM (CD31), vascularcell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule(ICAM)-1, a selectin such as P-Selectin (CD62P), E-Selectin (CD62E),L-selectin, and Flk-1, which also may be modified through bifunctionalantibody construct technology. In one embodiment, the linker is ananti-CD31 or anti-ICAM antibody attached to the therapeutic cell.

b. Genetic Modification

i. Transgenes

In one embodiment, a transgene is introduced into a therapeutic cell.The transgene encodes a gene product including but not limited to anadhesion molecule with an affinity for the luminal surface of the targetvasculature. Adhesion molecules include, for example, CD44, P-selectinglycoprotein ligand-1 (PSGL-1; CD 162), hematopoietic cell E-/L-selectinligand (HCELL), E-selectin ligand-1, Very Late Antigen-4 (VLA-4; CD49d),Leukocyte Function Associated Antigen-1 (LFA-1), an integrin, such as anα4 integrin or a β2 integrin, CD31, VE-Cadherin (CD144), PECAM (CD31),vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesionmolecule (ICAM)-1, a selectin such as P-Selectin (CD62P), E-Selectin(CD62E), L-selectin, α4β7, Mac-1, cutaneous lymphocyte antigen, CD34,CD133, VEGF receptor 1 (flt-1/flk-2), VEGF receptor 2 (flk-1/KDR), andCXCR4. The upregulation of one subset of these molecules enhancesadhesion of therapeutic cells to cells associated with a target site,for example, endothelial cells, by directly increasing the surfaceconcentration of adhesion sites, while another subset of these adhesionmolecules may require additional modification, as described herein, toenhance cell engraftment.

For purposes of the present subject matter, control elements, such aspromoters, enhancers and the like, will be of particular use. Suchcontrol elements include, for example, a cytomegalovirus promoter andvariants thereof (commercially available from Clontech or GenetherapySystems).

A transgenic therapeutic cell includes a transgene that enhances theengraftment, proliferation, survival, differentiation and/or function ofthe therapeutic cells and/or decreases, replaces or supplements(increases) the expression of endogenous genes in the therapeutic cells.In one embodiment, the expression of the transgene is controlled by aregulatable or tissue-specific, e.g., cardiomyocyte-specific promoter.Optionally, a combination of vectors each with a different transgene canbe employed.

(a) Exemplary Genes for Delivery

Exemplary genes for delivery to a therapeutic cell include those genesthat express adhesion molecules, as discussed herein.

(b) Delivery of Transgenes to Therapeutic Cells

Delivery of exogenous transgenes to a therapeutic cell may beaccomplished by any means, e.g., transfection with naked DNA, e.g., avector comprising the transgene, liposomes, association withpolycations, calcium-mediated transformation, electroporation, ortransduction, e.g., using recombinant viruses. A number of transfectiontechniques are generally known in the art. See, e.g., Graham et al.,Virology, 52, 456 (1973), Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratories, New York (1989),Davis et al., Basic Methods in Molecular Biology, Elsevier (1986) andChu et al., Gene, 13, 197 (1981). Particularly suitable transfectionmethods include calcium phosphate co-precipitation (Graham et al.,Virol., 52, 456 (1973)), direct microinjection into cultured cells(Capecchi, Cell, 22, 479 (1980)), electroporation (Shigekawa et al.,BioTechniques, 6, 742 (1988)), liposome-mediated gene transfer (Manninoet al., BioTechniques, 6, 682 (1988)), lipid-mediated transduction(Feigner et al., Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)), andnucleic acid delivery using high-velocity microprojectiles (Klein etal., Nature, 327, 70 (1987)).

An expression cassette optionally includes at least one control elementsuch as a promoter, for example, a commercially availablecytomegalovirus promoter, variants thereof, or optionally a regulatablepromoter, e.g., one which is inducible or repressible, an enhancer, or atranscription termination sequence. In certain embodiments, the promoterand/or enhancer is one which is cell- or tissue-specific, e.g., cardiaccell-specific. For instance, the enhancer may be a muscle creatinekinase (mck) enhancer, and the promoter may be an alpha-myosin heavychain (MyHC) or beta-MyHC promoter (see Palermo et al., Circ. Res., 78,504 (1996)).

In one embodiment, vectors are used to deliver exogenous transgenes totherapeutic cells. Vectors include, for example, viral vectors,liposomes and other lipid-containing complexes, and other macromolecularcomplexes capable of mediating delivery of a gene to a host cell.Vectors can also comprise other components or functionalities thatfurther modulate gene delivery and/or gene expression, or that otherwiseprovide beneficial properties to the targeted cells. Such othercomponents include, for example, components that influence binding ortargeting to cells (including components that mediate cell-type ortissue-specific binding); components that influence uptake of the vectorby the cell; components that influence localization of the transferredgene within the cell after uptake (such as agents mediating nuclearlocalization); and components that influence expression of the gene.Such components also might include markers, such as detectable and/orselectable markers that can be used to detect or select for cells thathave taken up and are expressing the nucleic acid delivered by thevector. Such components can be provided as a natural feature of thevector (such as the use of certain viral vectors that have components orfunctionalities mediating binding and uptake), or vectors can bemodified to provide such functionalities. Selectable markers can bepositive, negative or bifunctional. Positive selectable markers allowselection for cells carrying the marker, whereas negative selectablemarkers allow cells carrying the marker to be selectively eliminated. Avariety of such marker genes have been described, including bifunctional(i.e., positive/negative) markers (see e.g., WO 92/08796; and WO94/28143). Such marker genes can provide an added measure of controlthat can be advantageous in gene therapy contexts. A large variety ofsuch vectors are known in the art and are generally available.

Vectors include, but are not limited to, isolated nucleic acid, e.g.,plasmid-based vectors which may be extrachromosomally maintained andviral vectors, e.g., recombinant adenovirus, retrovirus, lentivirus,herpesvirus, including cytomegalovirus, poxvirus, papilloma virus, oradeno-associated virus (AAV), including viral and non-viral vectorswhich are present in liposomes, e.g., neutral or cationic liposomes,such as DOSPA/DOPE, DOGS/DOPE or DMRIE/DOPE liposomes, and/or associatedwith other molecules such as DNA-anti-DNA antibody-cationic lipid(DOTMA/DOPE) complexes. Exemplary viral vectors are described below.

Vectors are administered via intracoronary administration as describedherein, and transfer to cells may be enhanced using electroporationand/or ionophoresis.

Retroviral Vectors

Retroviral vectors exhibit several distinctive features including theirability to stably and precisely integrate into the host genome providinglong-term transgene expression. These vectors can be manipulated ex vivoto eliminate infectious gene particles to minimize the risk of systemicinfection and patient-to-patient transmission. Pseudotyped retroviralvectors can alter host cell tropism.

Lentiviruses

Lentiviruses are derived from a family of retroviruses that includehuman immunodeficiency virus and feline immunodeficiency virus. However,unlike retroviruses that only infect dividing cells, lentiviruses caninfect both dividing and nondividing cells. For instance, lentiviralvectors based on human immunodeficiency virus genome are capable ofefficient transduction of cardiac myocytes in vivo. Althoughlentiviruses have specific tropisms, pseudotyping the viral envelopewith vesicular stomatitis virus yields virus with a broader range(Schnepp et al., Meth. Mol. Med., 69:427 (2002)).

Adenoviral Vectors

Adenoviral vectors may be rendered replication-incompetent by deletingthe early (E1A and E1B) genes responsible for viral gene expression fromthe genome and are stably maintained into the host cells in anextrachromosomal form. These vectors have the ability to transfect bothreplicating and nonreplicating cells and, in particular, these vectorshave been shown to efficiently infect cardiac myocytes in vivo, e.g.,after direction injection or perfusion. Adenoviral vectors have beenshown to result in transient expression of therapeutic genes in vivo,peaking at 7 days and lasting approximately 4 weeks. The duration oftransgene expression may be improved in systems utilizing cardiacspecific promoters. In addition, adenoviral vectors can be produced atvery high titers, allowing efficient gene transfer with small volumes ofvirus.

Adeno-Associated Virus Vectors

Recombinant adeno-associated viruses (rAAV) are derived fromnonpathogenic parvoviruses, evoke essentially no cellular immuneresponse, and produce transgene expression lasting months in mostsystems. Moreover, like adenovirus, adeno-associated virus vectors alsohave the capability to infect replicating and nonreplicating cells andare believed to be nonpathogenic to humans. Moreover, they appearpromising for sustained cardiac gene transfer (Hoshijima et al., Nat.Med., 8:864 (2002); Lynch et al., Circ. Res., 80:197 (1997)).

In one embodiment, recombinant AAV (rAAV) is employed to deliver atransgene to therapeutic cells. Differentiation is induced by placingsubconfluent therapeutic cells in DMEM containing 2% horse serum andstandard concentrations of glutamine and penicillin-streptomycin for aninterval of four days prior to transduction.

Herpesvirus/Amplicon

Herpes simplex virus 1 (HSV-1) has a number of important characteristicsthat make it an important gene delivery vector in vivo. There are twotypes of HSV-1-based vectors: 1) those produced by inserting theexogenous genes into a backbone virus genome, and 2) HSV ampliconvirions that are produced by inserting the exogenous gene into anamplicon plasmid that is subsequently replicated and then packaged intovirion particles. HSV-1 can infect a wide variety of cells, bothdividing and nondividing, but has obviously strong tropism towards nervecells. It has a very large genome size and can accommodate very largetransgenes (>35 kb). Herpesvirus vectors are particularly useful fordelivery of large genes, e.g., genes encoding ryanodine receptors andtitin.

Plasmid DNA Vectors

Plasmid DNA is often referred to as “naked DNA” to indicate the absenceof a more elaborate packaging system. Direct injection of plasmid DNA tomyocardial cells in vivo has been accomplished. Plasmid-based vectorsare relatively nonimmunogenic and nonpathogenic, with the potential tostably integrate in the cellular genome, resulting in long-term geneexpression in postmitotic cells in vivo. For example, expression ofsecreted angiogenesis factors after muscle injection of plasmid DNA,despite relatively low levels of focal transgene expression, hasdemonstrated significant biologic effects in animal models and appearspromising clinically (Isner, Nature, 415:234 (2002)). Furthermore,plasmid DNA is rapidly degraded in the blood stream; therefore, thechange of transgene expression in distant organ systems in negligible.Plasmid DNA may be delivered to cells as part of a macromolecularcomplex, e.g., a liposome, polymer, e.g., cationic polymer, orDNA-protein complex, and delivery may be enhanced using techniquesincluding electroporation.

Regulatable Transcriptional Control Elements

A variety of strategies have been devised to control in vivo expressionof transferred genes and thus alter the pharmacokinetics of in vivo genetransfer vectors in the context of regulatable or inducible promoters.Many of these regulatable promoters use exogenously administered agentsto control transgene expression and some use the physiologic milieu tocontrol gene expression. Examples of the exogenous control promotersinclude the tetracycline-responsive promoter, a chimeric transactivatorconsisting of the DNA and tetracycline-binding domains from thebacterial tet repressor fused to the transactivation domain of herpessimplex virion protein 16 (Ho et al., Brain Res. Mol. Brain. Res.,41:200 (1996)); a chimeric promoter with multiple cyclic adenosinemonophosphate response elements superimposed on a minimal fragment ofthe 5′-flanking region of the cystic fibrosis transmembrane conductanceregulator gene (Suzuki et al., 7:1883 (1996)); the EGR1radiation-inducible promoter (Hallahan et al., Nat. Med., 1:786 (1995));and the chimeric GRE promoter (Lee et al., J. Thoracic Cardio. Surg.,118:26 (1996)), with 5 GREs from the rat tyrosine aminotransferase genein tandem with the insertion of Ad2 major late promoter TATAbox-initiation site (Narumi et al., Blood, 92:812 (1998)). Examples ofthe physiologic control of promoters include a chimera of the thymidinekinase promoter and the thyroid hormone and retinoic acid-responsiveelement responsive to both exogenous and endogenous tri-iodothyroniine(Hayashi et al., J. Biol. Chem., 269:23872 (1994)); complement factor 3and serum amyloid A3 promoters responsive to inflammatory stimuli; thegrp78 and BiP stress-inducible promoter, a glucose-regulated proteinthat is inducible through glucose deprivation, chronic anoxia, andacidic pH (Gazit et al., Cancer Res., 55:1660 (1995)); andhypoxia-inducible factor 1 and a heterodimeric basic helix-loop-helixprotein that activates transcription of the human erythropoietin gene inhypoxic cells, which has been shown to act as a regulatable promoter inthe context of gene therapy in vivo (Forsythe et al., Mol. Cell. Biol.,16:4604 (1996)).

Regulatable transcriptional elements include, but are not limited to, atruncated ligand binding domain of a progesterin receptor (controlled byantiprogestin), a tet promoter (controlled by tet and dox) (Dhawan etal., Somat. Cell. Mol. Genet., 21, 233 (1995); Gossen et al., Science,268: 1766 (1995); Gossen et al., Science, 89: 5547 (1992); Shockett etal., Proc. Natl. Acad. Sci. USA, 92, 6522 (1995)), hypoxia-induciblenuclear factors (Semenza et al., Proc. Natl. Acad. Sci. USA, 88, 5680(1991); Semenza et al., J. Biol. Chem., 269, 23757)), steroid-inducibleelements and promoters, such as the glucocorticoid response element(GRE) (Mader and White, Proc. Natl. Acad. Sci. USA, 90, 5603 (1993)),and the fusion consensus element for RU486 induction (Wang et al., Proc.Natl. Acad. Sci. USA, 91:818 (1994)), those sensitive to electromagneticfields, e.g., those present in metallothionein I or II, c-myc, and HSP70promoters (Lin et al., J. Cell. Biochem., 81: 143 (2001); Lin et al., J.Cell. Biochem., 54: 281 (1994); U.S. published application20020099026)), and electric pulses (Rubenstrunk et al., J. Gene Med.,5:773 (2003)), as well as a yeast GAL4/TATA promoter, auxin inducibleelement, an ecdysone responsive element (No et al., Proc. Natl. Acad.Sci. USA, 93:3346 (1996)), an element inducible by rapamycin (FK 506) oran analog thereof (Rivera et al., Nat. Med., 2:1028 (1996); Ye et al.,Science, 283:88 (1999); Rivera et al., Proc. Natl. Acad. Sci. USA,96:8657 (1999)), a tat responsive element, a metal, e.g., zinc,inducible element, a radiation inducible element, e.g., ionizingradiation has been used as the inducer of the promoter of the earlygrowth response gene (Erg-1) Hallahan et al., Nat. Med., 1:786 (1995)),an element which binds nuclear receptor PPARγ (peroxisome proliferatorsactivated receptors), which is composed of a minimal promoter fused toPPRE (PPAR responsive elements, see WO 00/78986), a cytochrome P450/A1promoter, a MDR-1 promoter, a promoter induced by specific cytokines(Varley et al., Nat. Biotech., 15:1002 (1997)), a light inducibleelement (Shimizu-Sato et al., Nat. Biotech., 20:1041 (2002)), a lacZpromoter, and a yeast Leu3 promoter.

In some embodiments, cell- or tissue-specific control elements, such asmuscle-specific and inducible promoters, enhancers and the like, will beof particular use, e.g., in conjunction with regulatable transcriptionalcontrol elements. Such control elements include, but are not limited to,those derived from the actin and myosin gene families, such as from themyoD gene family (Weintraub et al., Science, 251, 761 (1991)); themyocyte-specific enhancer binding factor MEF-2 (Cserjesi and Olson, Mol.Cell. Biol., 11, 4854 (1991)); control elements derived from the humanskeletal actin gene (Muscat et al., Mol. Cell. Biol., 7, 4089 (1987))and the cardiac actin gene; muscle creatine kinase sequence elements(Johnson et al., Mol. Cell. Biol., 9, 3393 (1989)) and the murinecreatine kinase enhancer (mCK) element; control elements derived fromthe skeletal fast-twitch troponin C gene, the slow-twitch cardiactroponin C gene and the slow-twitch troponin I genes.

Cardiac cell restricted promoters include but are not limited topromoters from the following genes: a α-myosin heavy chain gene, e.g., aventricular α-myosin heavy chain gene, β-myosin heavy chain gene, e.g.,a ventricular β-myosin heavy chain gene, myosin light chain 2v gene,e.g., a ventricular myosin light chain 2 gene, myosin light chain 2agene, e.g., a ventricular myosin light chain 2 gene,cardiomyocyte-restricted cardiac ankyrin repeat protein (CARP) gene,cardiac α-actin gene, cardiac m2 muscarinic acteylcholine gene, ANPgene, BNP gene, cardiac troponin C gene, cardiac troponin I gene,cardiac troponin T gene, cardiac sarcoplasmic reticulum Ca-ATPase gene,skeletal α-actin gene, as well as an artificial cardiac cell-specificpromoter.

Further, chamber-specific promoters or enhancers may also be employed,e.g., for atrial-specific expression, the quail slow myosin chain type 3(MyHC3) or ANP promoter, or the cGATA-6 enhancer, may be employed. Forventricle-specific expression, the iroquois homeobox gene may beemployed. Examples of ventricular myocyte-specific promoters include aventricular myosin light chain 2 promoter and a ventricular myosin heavychain promoter.

In other embodiments, disease-specific control elements may be employed.Thus, control elements from genes associated with a particular disease,including but not limited to any of the genes disclosed herein may beemployed.

Nevertheless, other promoters and/or enhancers which are not specificfor cardiac cells or muscle cells, e.g., RSV promoter, may be employed.Other sources for promoters and/or enhancers are promoters and enhancersfrom the Csx/NKX 2.5 gene, titin gene, α-actinin gene, myomesin gene, Mprotein gene, cardiac troponin T gene, RyR2 gene, Cx40 gene, and Cx43gene, as well as genes which bind Mef2, dHAND, GATA, CarG, E-box,Csx/NKX 2.5, or TGF-beta, or a combination thereof.

Targeted Vectors

The present subject matter contemplates the use of cell targeting notonly by delivery of the transgene or therapeutic cell into the coronaryartery, for example, but also by use of targeted vector constructshaving features that tend to target gene delivery and/or gene expressionto a particular host cells or host cell types (such as the myocardium).Such targeted vector constructs would thus include targeted deliveryvectors and/or targeted vectors, as described herein. Restrictingdelivery and/or expression can be beneficial as a means of furtherfocusing the potential effects of gene therapy. The potential usefulnessof further restricting delivery/expression depends in large part on thetype of vector being used and the method and place of introduction ofsuch vector. For instance, delivery of viral vectors via intracoronaryinjection to the myocardium has been observed to provide, in itself,highly targeted gene delivery. In addition, using vectors that do notresult in transgene integration into a replicon of the host cell (suchas adenovirus and numerous other vectors), cardiac myocytes are expectedto exhibit relatively long transgene expression since the cells do notundergo rapid turnover. In contrast, expression in more rapidly dividingcells would tend to be decreased by cell division and turnover. However,other means of limiting delivery and/or expression can also be employed,in addition to or in place of the illustrated delivery method, asdescribed herein.

Targeted delivery vectors include, for example, vectors (such asviruses, non-viral protein-based vectors, polymer-based and lipid-basedvectors) having surface components (such as a member of aligand-receptor pair, the other half of which is found on a host cell tobe targeted) or other features that mediate preferential binding and/orgene delivery to particular host cells or host cell types. As is knownin the art, a number of vectors of both viral and non-viral origin haveinherent properties facilitating such preferential binding and/or havebeen modified to effect preferential targeting (see, e.g., Miller, etal., FASEB Journal, 9:190 (1995); Chonn et al., Curr. Opin. Biotech.,6:698 (1995); Schofield et al., British Med. Bull., 51: 56 (1995);Schreier, Pharmaceutical Acta Helvetiae 68:145 (1994); Ledley, HumanGene Therapy, 6:1129 (1995); WO 95/34647; WO 95/28494; and WO96/000295).

Targeted vectors include vectors (such as viruses, non-viralprotein-based vectors and lipid-based vectors) in which delivery resultsin transgene expression that is relatively limited to particular hostcells or host cell types. For example, transgenes can be operably linkedto heterologous tissue-specific enhancers or promoters therebyrestricting expression to cells in that particular tissue. For example,tissue-specific transcriptional control sequences derived from a geneencoding left ventricular myosin light chain-2 (MLC₂V) or myosin heavychain (MHC) can be fused to a transgene within a vector. Expression ofthe transgene can therefore be relatively restricted to ventricularcardiac myocytes.

Additional gene transfer methods are also contemplated, such aspackaging of DNA with polycations into nanoparticles. Positively chargedpolycations complex spontaneously with DNA, which is negatively charged,resulting in self-assembled nanoparticles. If the DNA charge isover-compensated, the resulting particle charge is positive, which thendrives the association with negatively charged cell membranes, therebyfacilitating transfection. As an example of gene delivery using apolycation, see Sweeney et al., Cancer Research, 63: 4017-4020 (2003).Exemplary polycations include, but is not limited to, polylysine,polyethylenimine, such as in vivo-jetPEI™ (Avanti® Polar Lipids, Inc.)and protamine.

c. Exemplary Methods to Characterize the Phenotype of Therapeutic CellsSubjected to Biological Conditioning

Methods to detect expression of a transgene in a therapeutic cellinclude methods that detect transgene-specific RNA, e.g., RT-PCR, ormethods that detect a gene product encoded by the transgene, e.g., viaan ELISA. Examples of gene-specific assays include, for instance, thosefor AC (see, Salomon et al., Anal. Biochem., 58, 541 (1974); Hammond etal., Circulation, 85, 269 (1992); Hammond et al., Circulation, 8, 666(1992)), for β-adrenergic receptor binding or content (Hammond et al.,Circulation, 8, 666 (1992); Roth et al., FEBS Lett., 29, 46 (1992)), forGRK₂ and GRK₅ content (see, e.g., Ping et al., J. Clin. Invest., 95,1271 (1995); and Roth et al., FEBS Lett, 29, 46 (1992)), and for Gprotein receptor kinase activity (see, Benovic, Methods Enzymology, 200,351 (1991); Ping et al., J. Clin. Invest., 95, 1271 (1995); Ping et al.,J. Clin. Invest., 95,1271 (1995); Ungerer et al., Circulation, 87, 454,(1993)).

In one embodiment, therapeutic cells are cardiomycytes, e.g., preparedfrom cardiac tissue or noncardiac tissue. Detection of expression ofcardiomyocyte-specific proteins may be accomplished using antibodies to,for example, myosin heavy chain monoclonal antibody, e.g., MF 20 (MF20),sarcoplasmic reticulum calcium ATPase (SERCAI), e.g., mnAb 10D1, or gapjunctions, e.g., using antibodies to connexin 43, as well asphospholamban, or by detecting the expression of the following genes:titin (Z-band), α-actinin, myomesin, sarcomeric myosin heavy chain,sarcomeric α-actin, cardiac tropinin T, M protein, RyR2, Cx40 and Cx 43.For the differentiation of ES cells to cardiomyocytes, the expression ofthe following genes may be monitored: Nkx 2.5, MEF2c, GATA 4/5/6,desmin, M-cadherin, beta1-integrin, oxytocin, oxytocin receptor, cardiacmyosin heavy chain, myosin light chain 2A or 2C, cardiac tropinin I,troponin C and ANP. For the differentiation of bone marrow derived MSCs,the expression of the following genes may be monitored: beta1 and beta2adrenergic receptors, e.g., via the response of cells to isoproterenol,or muscarinic receptors, e.g., via the response of cells to carbachol.

Atrial-like cells may be identified as cells having ion currentsassociated with muscarinic acetylcholine-activated K⁺ channels andinwardly rectifying K⁺ channels, but not hyperpolarization-activatedpacemaker channels, while ventricular-like cells may be identified ascells having ion currents associated with inwardly rectifying K⁺channels and SR ryanodine-sensitive calcium-release channels but notmuscarinic acetylcholine-activated K⁺ channels orhyperpolarization-activated pacemaker channels. Sinus node-like cellsmay be identified as cells having ion currents associated withmuscarinic acetylcholine-activated K⁺ channels and SRryanodine-sensitive calcium release channels, andhyperpolarization-activated pacemaker channels but not inwardlyrectifying K⁺ channels.

3. Chemical Conditioning

In another embodiment, molecules or molecular moieties possessingaffinity to the luminal surface of the target area vasculature arechemically conjugated to the surface of a therapeutic cell using methodsknown to the art, as illustrated in FIGS. 2A and 2B. FIG. 2A illustratessurface modification of therapeutic cells using NHS reactive linkermolecules. In the figure, the NHS linker molecules each include NHS andanti-E. FIG. 2B illustrates a therapeutic cell attached to anendothelial cell of the target vasculature. The molecule or molecularmoiety may be conjugated to the cell surface via a spacer molecule toenhance accessibility. One such molecule may possess more than onemolecular moiety with affinity to the target surface. In certainembodiments, the spacer may be branched. Attachment molecules may bechemically conjugated, i) to amine groups using reactive esters,epoxide, aldehydes, ii) to sulfhydryl groups using maleimides, vinylsulfones, iii) to carboxyl groups using dimethylaminopropyl-carbodiimide(EDC) chemistry, iv) or non-selectively using photochemistry.

For example, the surface of the therapeutic cell, e.g., a stem cell, maybe modified to display or express an antibody to a receptor present onan endothelial cell of the target vasculature, e.g., E-Selectin, PECAM(CD31), and the like. In addition to the biological conditioningmethodology as described herein, the therapeutic cell may be modified bychemically conjugating a vinyl sulfone (VS)-PEG-antibody molecule tosulfhydril groups to the cell surface. To generate a VS-PEG-antibodymolecular construct, a cysteine residue may be inserted in the Cterminus of the antibody or antibody fragment by using geneticengineering methodologies. The genetic code of an antibody of interestmay be obtained, for example, from clonal selection through phagedisplay. The genetic code of a monoclonal antibody may be modified toinclude a cysteine residue at the C terminus and expressed in abacterial or mammalian expression system (Härmä et al., ClinicalChemistry, 46:1755-1761 (2000)). These engineered antibodies may beincubated with a molar excess of VS-PEG-VS to yield the abovesulfhydril-reactive VS-PEG-antibody.

Alternatively, antibodies may be attached to a therapeutic cell surfacethrough a linking bridge, e.g., a biotin-avidin bridge. For thispurpose, NHS-PEG-biotin is conjugated to the cells by incubating cellswith the NHS-PEG-biotin. Subsequently, cells are incubated with avidin,or derivatives thereof such as streptavidin, NeutrAvidin and the like.As avidin provides four opposing (two on each side) binding pockets forbiotin, the cell surface will present two empty avidin pockets on itssurface. In a final step, biotinylated antibodies are attached to thecell surface by incubation of the biotinylated antibody with cellspresenting avidin at their surface

As above, other antibodies, fragments thereof, or molecules other thanantibodies may be conjugated to the surface of a therapeutic cell.

Molecules or molecular moieties possessing affinity to the luminalsurface of the target area vasculature may be introduced into andanchored in the membrane of a therapeutic cell by liposomal or micelledelivery.

For example, CD31 antibodies or fragments thereof may be conjugated to aphosphatidyl ethanolamine lipid with di-C16 or longer chains. The lipidanchor may than be introduced into the cell membrane through micelle orliposomal fusion. Alternatively, hydrophobic peptide alpha-helices (suchas poly-leucine), short or hydrophobic polymer chains may serve asmembrane anchors.

As above, other antibodies, fragments thereof, or molecules other thanantibodies, e.g., affibodies, may be anchored in the membrane of thetherapeutic cells. For molecules with an inherent transmembrane-domain,modification may not be necessary.

Proteins and peptides are amino acid polymers containing a number ofreactive side chains. In addition to, or as an alternative to, theseintrinsic reactive groups, specific reactive moieties can be introducedinto a polymer chain by chemical modification. These groups, whether ornot they are naturally a part of the protein or are artificiallyintroduced, serve as “handles” for attaching a wide variety ofmolecules, including other proteins. The intrinsic reactive groups ofproteins are described in the following section.

(a) Amines (Lysines, α-Amino Groups). One of the most common reactivegroups of proteins is the aliphatic ε-amine of the amino acid lysine.Lysines are usually present to some extent and are often quite abundant.Lysine amines are nucleophiles above pH 8.0 (pK_(a)=9.18) and thereforereact with a variety of reagents to form stable bonds. Other reactiveamines that are found in proteins are the α-amino groups of theN-terminal amino acids. The α-amino groups are less basic than lysines,are reactive at around pH 7.0, and can be selectively modified in thepresence of lysines.

(b) Thiols (Cystine, Cysteine, Methionine). Another common reactivegroup in proteins is the thiol residue from the sulfur-containing aminoacid cysteine and its reduction product cysteine (of half-cystine),which are counted together as one of the 20 amino acids. Cysteinecontains a free thiol group, which is more nucleophilic than amines andis generally the most reactive functional group in a protein. It reactswith some of the same modification reagents as do the amines discussedin the previous section and in addition can react with reagents that arenot very reactive towards amines. Thiols, unlike most amines, arereactive at neutral pH, and therefore they can be coupled to othermolecules selectively in the presence of amines. This selectivity makesthe thiol group the linker of choice for coupling two proteins together,since methods that only couple amines (e.g., glutaraldehyde,dimethyladipimidate coupling) can result in formation of homodimers,oligomers, and other unwanted products. Since free sulfhydryl groups arerelatively reactive, proteins with these groups often exist in theiroxidized form as disulfide-linked oligomers or have internally bridgeddisulfide groups. Immunoglobulin M is an example of a disulfide-linkedpentamer, while immunoglobulin G is an example of a protein withinternal disulfide bridges bonding the subunits together. In proteinssuch as this, reduction of the disulfide bonds with a reagent such asdithiothreitol (DTT) is required to generate the reactive free thiol. Inaddition to cystine and cysteine, some proteins also have the amino acidmethionine, which contains sulfur in a thioether linkage. When cysteineis absent, methionine can sometimes react with thiol-reactive reagentssuch as iodoacetamides.

(c) Phenios (Tyrosine). The phenolic substituent of the amino acidtyrosine can react in two ways. The phenolic hydroxyl group can formesters and ether bonds, and the aromatic ring can undergo nitration orcoupling reactions with reagents such as diazonium salts at the positionadjacent to the hydroxyl group. Tyrosyl residues can react withdiazonium compounds. For example, a p-aminobenzoyl biocytin derivativehas been diazotized and reacted with protein tyrosine groups.

(d) Carboxylic Acids (Aspartic Acid, Glutamic Acid). Proteins containcarboxylic acid groups at the carboxy-terminal position and within theside chains of the dicarboxylic amino acids aspartic acid and glutamicacid. The low reactivity of carboxylic acids in water usually makes itdifficult to use these groups to selectively modify proteins and otherbiopolymers. In the cases where this is done, the carboxylic acid groupis usually converted to a reactive ester by use of a water-solublecarbodiimide and then reacted with a nucleophilic reagent such as anamine or a hydrazide. The amine reagent should be weakly basic in orderto react specifically with the activated carboxylic acid in the presenceof the other amines on the protein. This is because proteincross-linking can occur when the pH is raised to above 8.0, the rangewhere the protein amines are partially unprotonated and reactive. Forthis reason, hydrazides, which are weakly basic, are useful in couplingreactions with a carboxylic acid. This reaction can also be usedeffectively to modify the carboxy terminal group of small peptides.

(e) Other Amino Acid Side Chains (Arginine, Histidine, Tryptophan). Thechemical modification of other amino acid side chains in proteins hasnot been extensive, compared to the groups discussed above. The highpK_(a) of the guanidine functional group of arginine (pK_(a)=12-13)necessitates more drastic reaction conditions than most proteins cansurvive. Arginine modification has been accomplished primarily withglyoxals and α-diketone reagents. Tryptophan modification requires harshconditions and is seldom carried out except as a method of analysis instructural or activity studies. Histidines have been subjected tophotooxidation and reaction with iodoacetates.

(f) Non-specific attachment. Photoreactive chemistry may be used toattach molecules to the cell surface in a non-specific way. Whenactivated by UV or visible radiation, photoreactive groups react withcarbohydrates, proteins or lipids present at the cell membraneinterface. Examples for photoreactive moieties include but are notlimited to phenyl azides, nitrophenyl azides, hydroxyphenyl azides.

(g) Other Methods.

In another embodiment, irritants and/or stimulants may be mixed withtherapeutic cells immediately before infusion.

As discussed herein, the surface of a therapeutic cell is modified toenhance engraftment of the cell at a target vasculature site or region.In one embodiment, “receptor-ligand” interaction is exploited to enhanceengraftment. For example, the HCELL adhesion molecule present on atherapeutic cells interacts with E-Selectin present on an endothelialcell at the target site, PSGL interacts with P-Selectin, and VLA-4interacts with VCAM-1 and/or ICAM-1. Thus, the upregulation of any ofthese adhesion molecules, or the introduction of any of these adhesionmolecules onto the respective cell membranes, by any methodology asdiscussed herein, will increase the membrane concentration of thesemolecules and therefore, increase the affinity between the therapeuticcell and target cell. In one embodiment, either the expression, display,or both of HCELL, E-Selectin or both is either upregulated, increased,or both, because of E-Selectin's involvement in the initial recruitmentand initiation of rolling adhesion.

In another embodiment, any combination of the methods discussed hereinmay be used to enhance engraftment of a therapeutic cell to a targetcell For example, the expression of CD34 on a stem cell can beupregulated by the genetic methods discussed herein, and thensubsequently modified with a bi-functional linker molecule as discussedto enhance engraftment of the stem cell at target endothelium.

D. Delivery and Infusion Regimes of To Enhance Engraftment ofTherapeutic Cells

As disclosed herein, the present subject matter is directed to anapparatus and method to enhance the engraftment of therapeutic cells ata target site in vasculature. To increase therapeutic cell attachment atthe target site, therapeutic cells can be conditioned mechanically toupregulate the expression of adhesion molecules. In another embodiment,the engraftment of therapeutic cells at the site of target vasculatureis enhanced by regulating the hemodynamics of the delivered therapeuticcell solution to establish flow dynamics conducive to therapeuticcell/target site interaction. For example, therapeutic cell residencytime at the target site can be increased by increasing the viscosity ofthe therapeutic cell solution to reduce the flow rate of the cellsolution, by impeding the flow proximally by employing flow resistance,or by proximal occlusion, which allows for control of the flow rate ofthe infused cell suspension.

1. Exemplary Protocols for Shear-Induced Modification of Cell Surface

Numerous cell types are known to be activated by shear at shear stressrates above 120 dynes/cm2 (Moritz et al., Thrombosis Research,22:445-455 (1981)). In one embodiment, therapeutic cells are loaded intoa catheter. With the tip of a catheter immersed in the cell suspension,a syringe pump is programmed to perform 1-3 inject/withdraw cycles athigh shear rates of, for example, 80 dynes/cm² and above.

Suitable syringe pumps for use in the present invention includecommercially available syringe pumps such as bench top models (New EraPump Systems, Inc., Farmingdale, N.Y., USA, www.syringepump.com; TedPella, Inc., Redding, Calif., USA, www.tedpella.com). U.S. Pat. No.5,342,298 discloses a programmable pump to deliver cells through aninfusion catheter into myocardium. The cells in the catheter will thenbe activated, and, as discussed herein, adhesion molecules will beupregulated and expressed. Once the cells are delivered into the targetvasculature, they will be able to more quickly adhere to targetvasculature and extravasate into the surrounding myocardium.

In an alternative embodiment, therapeutic cells are shear activatedbefore loading into a catheter, for example, by controlled shaking oragitation in a table-top device.

In another embodiment, the inject/withdraw cycles may be performed withthe catheter already positioned at the delivery site and with occlusionballoon on. In this case, the shear cycle may not only activate thetherapeutic cells that are in the delivery system, but may alsoupregulate adhesion molecules on the endothelial cells lining. This willallow better adhesion of the delivered cells, e.g., bone marrowmononuclear fraction, to the vasculature that contains endothelialcells.

In yet another embodiment, the inject/withdraw cycle may be performedwith the catheter positioned at the delivery site and with occlusionballoon on. Shear activation is performed with a saline flush toactivate target endothelial cells only. The therapeutic cells are thendelivered to the target vasculature post saline flush.

In one embodiment, the shear rate targets are defined for eachindividual cycle. Shear rate is a function of length of the catheterlumen in which cells are residing, as well as the diameter of suchcatheter, and fluid velocity produced by the syringe. The followingformula may be used:

Tau(shear stress)=−mu(dv/dr) and Tau=(delP/2L)r

With these two equations, sheer stress one can solve for (Tau) usingknown device (R, L), pump (Del P and DV/dr), and fluid (mu) parameters.[Tau=Fluid Shear Stress; mu=Fluid Viscosity; V=Fluid Velocity (which maybe selected on a syringe pump, and is function of the syringe size);r=Radial Distance (distance from the center of the circularcross-section or inner lumen radium); P=pressure generated by the pumpand L is length of the tube]. Bird et al., Transport Phenomena (1960).

2. Protocols to Induce Proximal Flow Impedance

The residence time of therapeutic cells, i.e., the duration of time thetherapeutic cells are in the vicinity of the target site or area, can beincreased to enhance retention at target site by controllinghemodynamics, for example, by reducing the flow rate in the target areavasculature. The flow rate can be controlled using a proximal flowimpedance device. Exemplary impedance devices include a partiallyinflated balloon, a doughnut-shaped balloon (having a middle openingthat provides reduced blood flow rate), a balloon with longitudinalchannels in the surface, a spiral balloon (having profusions at the timeof some delivery and wherein the flow is reduced by forcing migrationbetween adjacent spirals) or a balloon having fluid flow constrictions.In one example, flow can be temporarily stopped by complete flowocclusion immediately after infusion of therapeutic cells and at a timewhen approximately a large number of therapeutic cells are locatedwithin the target area.

In another example, a proximal flow impedance device is introduced toinduce ischemia. According to one example, the blood flow is notentirely occluded but rather, merely reduced to some non-zero flow rate.The non-zero flow rate allows infusion of therapeutic cells. In oneexample, the flow is reduced from an initial flow rate by a factor of 10to 70 percent. Typically, an arterial occlusion is considered clinicallyrelevant or flow limiting when the occlusion is approximately 70% orgreater. Accordingly, the level of occlusion provided by a flow resistorof the present subject matter will by approximately 70-100% of normalvessel diameter. As noted, exemplary flow resistors include a balloonhaving longitudinal grooves along the perimeter through which fluidflows or a doughnut-shaped balloon in a manner similar to a vessellesion.

In one example, the residence time is increased by changing thehemodynamics of the therapeutic cells. The hemodynamics can be tailoredby increasing the viscosity of the solution causing the solution to flowmore slowly. Increasing the viscosity serves to impede the flow proximalto the target site. In addition, flow proximal to the target site can bereduced by adding a flow resistance.

Other kinds of flow resistors are also contemplated, including, forexample, an insertion device fabricated of porous or sintered material.

3. Delivery of Viscous Agents to Enhance Engraftment of TherapeuticCells

Once delivered to the capillary bed, a therapeutic cell contacts a cellat the target site, e.g., an endothelial cell. If engaging theendothelial cell through focal molecular adhesion (receptor to ligandbinding), the therapeutic cell rolls along the endothelial surface untilit either breaks free or adheres firmly. An example for such rollinginteraction is the rolling of leucocytes along endothelial cells.Rolling speeds are on the order of 40-60 microns/sec, (Ramos, C. et al.,Circ Res., 84: 1237-1244 (1999); Prorock, A. et al., Am J Physiol HeartCirc Physiol., 284: H133-H140 (2003); Baudry, N. et al., Am J RespirCrit. Care Med, 158: 477-483 (1998)) which translates into a contacttime between a rolling cell and a given endothelial cell of roughly 0.5seconds.

To increase the probability of a therapeutic cell contacting a targetcell, in one embodiment, viscous agents are delivered with thetherapeutic cells to a subject. Factors that effect blood viscosityinclude plasma viscosity, aggregation of red blood cells, internalviscosity of red cells, hemoconcentration, aggregation of platelets andconcentration of white cells. Flow velocity is inversely proportional toviscosity. Higher viscosity leads to a reduced rate of flow, which inturn increases the residence time of the therapeutic cells at the targetsite, thus increasing the time in which the therapeutic cells have toengraft, i.e., adhere and transmigrate into the target site, e.g., aninfarcted region.

For example, adding a sufficient amount (about 0.25%-5% by weight) of ahigher viscosity biobeneficial/biocompatible medium such as tocopherol(Vitamin E), lipid emulsions such as emulsified vegetable oil,surfactant (Cremaphor), or a hydrophilic polymer increases the viscosityof the plasma or the therapeutic cell injection medium, thus increasingthe time of residence of therapeutic cells at the target site duringcell delivery. Examples of suitable hydrophilic polymers are PEG, PVA(Polyvinyl alcohol), PVP (polyvinylpyrrolidone), Dextran, and dextransulfate. Molecular weights of the dissolved hydrophilic polymers canrange up to 200K Daltons, and in one embodiment are between 5K to 30KDaltons. These higher viscosity biobeneficial/biocompatible mediaincrease the viscosity of plasma, thus increasing the time of residenceof therapeutic cells at the target site during cell delivery.

Alternatively, to increase the residency time of the therapeutic cell ata target location, the therapeutic cell is contacted with, e.g.,incubated with, activated platelets or platelet-derived microparticlesto cause the formation of clumps or rosettes. (Janowska-Wieczorek etal., Blood, 98:3143-3149 (2001)). After filtering/controlling the clumpsize, a composition containing well controlled therapeutic cell clumpsleads to embolization in the capillaries, which increases the dwell timeof the cells in the capillaries as well as provides a transient ischemicepisode that upregulates adhesion and homing molecules in thesurrounding interstitial space. Thus, an efficient transport oftherapeutic cells to the target site is provided.

In other embodiments, an engraftment enhancing agent such as a calciumionophore, oleic acid, histamine, DMSO, histamine, bradykinin,serotonin, thrombin, VEGF, a leukotriene such as LTC4, LTD4, LTE4, or avasodilator, such as an ACE inhibitor or a nitrate, are added to theinjectate to open the interstitial spaces and/or increase vascular wallpermeability for more effective cell delivery (Rutledge et al.,Circulation Research, 66: 486-495 (1990); Saxena et al., J. Clin.Invest., 89: 373-380 (1992); Gupta et al., J. Leukoc Biol., 70(3):431-438 (2001); and van Nieuw Amerongen et al., Circ. Res., 83:1115-1123(1998)). Liu et al., Am. J. Hematol., 74: 216-217 (2003) reported thatadhesion molecules were dramatically increased on CD34⁺ cells surface inthe presence of platelet microparticles, and these cells were shown toadhere better on endothelial cells and fibronectin.

In an alternative embodiment, therapeutic cells, e.g., stem cells, aredelivered in a two-phase process via the capillaries leading to thetarget site, e.g., myocardial tissue of the heart, by means of thedelivery device described herein. Phase One (1) includes the use of ahigh viscosity “foam” that contains CO₂ and an engraftment enhancingagent such as a cytokine, e.g., oxidized LDL, tumor necrosisfactor-alpha, interleukin-1 and other cytokines that stimulate theexpression of cell adhesion molecules on the surfaces of cells, and/or achemokines, such as IL-8, SDF-1, MIP-1, MCP-1/2/3/4 and lymphoactin. Inaddition, a device such as the one disclosed in Gordilo et al., Physicsof Fluids, 16: 2828 (2004) can be used to generate the CO₂ microbubbles.

In another embodiment, the “foam” includes ultrasonic contrast agentsand CO₂ microbubbles. Several cardiac and intravenously injectablevascular ultrasound contrast agents are commercially available, such asAlbunex (Molecular Biosystems), Optison (Molecular Biosystems), Echovist(Schering), Levovist (Schering), EchoGen (Sonus Pharmaceuticals),Definity (Du Pont Merck), Imagent (Alliance Pharmaceutical), Sonazoid(Nycomed-Amersham), SonoVue (Bracco Diagnostics), Quantison (Quadrant),Biosphere (Ponit Biomedical), and AI-700 (Acusphere).

The physical nature of the solution results from the use of a mixingdevice, for example, associated with the delivery device. The CO₂microbubbles promote tissue ischemia (necessary ischemicpreconditioning) by elevating the concentration of carbon dioxide in theblood. This elevation decreases the concentration gradient for carbondioxide between the cardiac cells and the blood resulting in a decreaseddiffusion of carbon dioxide out of the cardiac cells and an elevatedcarbon dioxide level inside the cells. The elevated carbon dioxide levelinduces an ischemic state with an decrease tissue pH (acidosis). Theengraftment enhancing agent induces a firm adhesion of the therapeuticcells to the capillary walls once the cells are introduced in Phase Two(2). Phase Two optionally includes the application of high viscosity“foam” containing CO₂ and therapeutic cells mixed together. By “foam” ismeant a collection of CO₂ microbubbles of sufficient size to increaseblood viscosity and slow the movement of blood and the cells therein asthey pass through capillaries and venules. Both the premixed engraftmentenhancing agent solution and the premixed therapeutic cell solution arerelatively high in viscosity, compared to blood viscosity. The viscousnature of the foam combined with the microbubbles of CO₂ would allow thetherapeutic cells to remain in the particular area for a predeterminedperiod of time. Once the CO₂ is absorbed, the foam dissipates, resumingnormal blood flow through the myocardia.

In one embodiment, an additional agent is delivered via the device toneutralize the foam.

4. Delivery of Agents to Increase ‘Bumping’ Frequency

The probability of a therapeutic cell successfully engrafting at atarget site increases if platelets are added to cause more bumping ofthe cells against the vessel surface. The process of cell extravasationfrom the vessel wall into the myocardium involves the upregulation ofadhesion molecules on the cell surface, as well as on the surface of thevessel's endothelium layer, and the ability of a therapeutic cell tointeract with the endothelium. Thus, in order to initiate cell/surfacecontact, a therapeutic cell must first “bump” into the adherent surface,then roll along the surface at a velocity slow enough to allow adhesionmolecules present on the therapeutic cell to contact, e.g., bind, with acorresponding adhesion molecules present on the target cell. Thefrequency of “bumping” dictates the number of therapeutic cells thatwill adhere to the endothelium and extravasate. For example, it has beenreported that leukocytes in close proximity to endothelial cells uponentry into postcapillary venules experience frequent collisions witherythrocytes, which pushes the cells towards the vessel wall (Stein etal., The Journal of Experimental Medicine, 189: 37-39 (1999)).

In one embodiment, microbubbles with sizes equivalent to red blood cellsare administered together with the therapeutic cell solution to increasethe collision frequency potential, which optimizes the chance ofcellular adhesion. In one embodiment, the microbubbles are about 10microns in diameter, and are composed of a lipid such as phospatidylcholine, albumin, a degradable polymer such as polycaprolactone, PLGApoly(lactide-co-glycolide), Polyester-amide, polyphosphazine, tyrosinecarbonate, and the like, or any combination thereof. The addition ofmicrobubbles to the therapeutic cell solution increases the apparentviscosity of the fluid, which slows down the rolling of the therapeuticcells. In another embodiment, the microbubbles serve as a carrier of asubstance, such as oxygen or NO to either induce a transient, localischemic environment or to provide more oxygenation in the event ofprolonged ischemia.

In another embodiment, platelet-derived microparticles (PMPs) areemployed to increase bumping frequency. PMPs are released uponactivation of platelets and express functional adhesion receptors,including αIIbβ3 (CD41), P-selectin (CD62P), and other platelet membranereceptors such as CXCR4 and PAR-1 (Janowska-Wieczorek et al., Blood,98:3143-3149 (2001)). PMPs released by activated platelets bind tomembranes of therapeutic cells, e.g., CD34⁺ therapeutic cells, andincrease their adhesion to endothelial cells (Id.). PMPs can be preparedusing methods known to the art. For example, PMPs can be collected fromblood by centrifugation. Briefly, blood is centrifuged in order tocollect platelet-rich plasma. Platelet rich plasma is then centrifugedin order to obtain platelet poor plasma. The platelet poor plasma isthen centrifuged to collect the platelet microparticles (PMPs). In oneembodiment, PMPs are collected from aged blood. See, for example, Forlowet al., Blood, 95: 1317-1323 (2000); Janowska-Wieczorek et al., Blood,98: 3143-3149 (2001).

Bumping frequency can also be increased by employing liposomes, lipidvesicles or vesicles with membranes formed from di-block or tri-blockco-polymers that can increase the viscosity of the medium and bumpingfrequency of the therapeutic cells with the endothelium when added tothe delivery medium. The liposomes, lipid vesicles or vesicles withmembranes formed from di-block or tri-block co-polymers have a size ofapproximately 100 nm-20 μm diameter, for example, approximately 100 nm-1μm diameter. In one embodiment, the co-polymers have a sizeapproximately 3-15 μm. In one embodiment, the liposome includes atherapeutic agent.

In yet another embodiment, microspheres are employed to increase bumpingfrequency. The microspheres may be composed of degradable polymers suchas polycaprolactone, PLGA poly(lactide-co-glycolide), Polyester-amide,polyphosphazine, tyrosine carbonate, etc., or Alginate crosslinked withdivalent Ca, Ba or Sr cations. Microspheres may also be made of anextra-cellular matrix protein such as collagen or gelatin, crosslinkedwith glutaraldehyde to prevent quick dissolution.

5. Delivery of Agents to Enhance Homing of Therapeutic Cells to TargetVasculature

Stem cells are precursor cells capable of proliferation, self-renewal,and differentiation into specialized tissues and organs, includingcardiomyocytes. The repopulation of cardiomyocytes to regenerate newmyocardium can mitigate the remodeling process. The “homing process”involves stem cell migration to the sites of injury or ischemia, whichprovides an environment that is favorable to growth and function. Thismicroenvironment is a stimulus for homing and differentiation of stemcells of the appropriate lineage. It increases vascular permeability andexpression of adhesion proteins like integrin, along with homingreceptors that facilitate their attachment, which is mediated bycell-to-cell contact and chemoattractant release from local tissueinjury.

In one embodiment, chemokines, a superfamily of small proteins thatfunction as potent chemotactic agents, some of which have a tissue- andinflammation-specific distribution, and others which are widelydistributed, are exploited to attract the therapeutic cells to thetarget vascular endothelium. For example, SDF-1 plays a role in homing(Sackstein, J. Invest. Dermatol., 122: 1061-1069 (2004)). SDF-1 isinfused into the target vasculature before infusion of therapeutic cellsto provide a homing stimulus. Alternatively, SDF-1 is contacted withtherapeutic cells prior to infusion, which contact initiates homingsignaling pathways leading to increased retention of therapeutic cellsupon infusion into the target vasculature.

Alternatively, it has been shown that exposure of cell to cytokinesalters expression patterns of these cells. Therefore, infusion ofplatelet derived cytokines/growth factors, VEGF, FGF, prior to infusionof therapeutic cells may increase cell retention at target vasculature.

Cytokines, such as granulocyte colony-stimulating factor (G-CSF) andstem cell factor (SCF), increase bone marrow stem cell mobilization,homing, and engraftment to infarcted myocardium. The endogenous repairprocess after myocardial necrosis can also be enhanced with specificgrowth factors, such as insulin-like and hepatocyte growth factors, thatstimulate cardiomyocyte replication and attract cardiac resident stemcells.

The migratory capacity of transplanted progenitor cells might bedependent on natural growth factors such as vascular endothelial growthfactor (VEGF) and stromal cell-derived factor-1 (SDF-1). The expressionof VEGF and SDF-1 is highly up-regulated in hypoxic tissue, supportingthe hypothesis that these factors may represent homing signals crucialto the recruitment of circulating progenitor cells to assist theendogenous repair mechanisms in the infarcted tissue.

Transplanted stem cells must engraft and proliferate efficiently aftermyocardial infarction to derive a maximal clinical benefit. With asmooth transition process, newly formed cardiomyocytes are required tobe connected intercellularly through electrical coupling with othercardiomyocytes and the formation of connexin, an integral membraneprotein constituent of gap junctions. Paramount to the survival of thestem cells is simultaneous neovascularization to keep up with themetabolic requirements of the newly transplanted cells to performcontractile work.

As described herein, mediators of stem cell mobilization, migration andattachment include granulocyte colony-stimulating factor, stem cellfactor, vascular endothelial growth factor (VEGF) and stromalcell-derived factor-1 (SDF-1).

In another embodiment, magnetically targeted therapy is used tomanipulate the homing process. Therapeutic cells, such as stem andprogenitor cells including hematopoeitic progenitor (CD34⁺) andmesenchymal stem cells (MSCs), take up and incorporate into perinuclearendosomes micron-scale iron oxide particles without affecting cellproliferation or functional capabilities (Hinds et al., Hematopoiesis,102: 867-872 (2003)). Therapeutic cells are contacted with suchparticles, which are either attached to the cell or taken up by thecell, and delivered to a subject. As described herein, application of amagnetic field gradient following delivery of the therapeutic cells andmagnetic carriers enhances engraftment of therapeutic cells to thetarget vasculature (see in general Pankhurst et al., J. Phys. D: Appl.Phys., 36: R167-R181 (2003)). Suitable magnetic particles are known tothe art, and include super-paramagnetic nanoparticles with iron oxide.Additional examples of suitable particles are shown in Tables 1 and 2.

TABLE 1 Polymer End composition/ groups and Other Diameter surfaceactivation Immobilized immobilized Name (μm) modification possibilityantibodies compounds Manufacturer/supplier BioMag −1 Silanization of—COOH, Secondary Abs, Protein A, PerSeptive Biosystems, iron oxides —NH₂anti-CD Abs, protein G, Farmingham, MA, USA anti-fluoresceinstreptavidin, Ab biotin Dynabeads M-280 2.8 Polystyrene Tosyl- SecondaryAbs, Streptavidin, Dynal, Oslo, Norway Dynabeads M-450 4.5 activatedanti-CD Abs, oligo (dT) Dynabeads M-500 5 Abs against E. coli O157,Salmonella Listeria, Cryptosporidivan Estapor −1 Polystyrene —COOH,Prolabo, Fontenay-sous-Bois, —NH₂ France Iobeads −1 Anti-CD Abs, AvidinImmunotech, Marseille, secondary Abs France M 100 1-10 Cellulose —OHScigen, Sittingbourne, UK M 104 M 108 MagaBeads 3.2 Polystyrene —COOH,Secondary Abs Streptavidin Cortex Biochem., San —NH₂, protein A,Leandro, CA, USA epoxy protein G Magne-Sphere <1 Streptavidin Promega,Madison, WI, USA Magnetic beads 0.8 Latex Streptavidin, ProZyme, SanLeandro, CA, protein A, USA protein G Magnetic 1-2  Polystyrene —COOHProtein A Polysciences, Warrington, microparticles —NH₂ PA, USA Magneticparticles 1 Polystyrene Anti-digoxigenin Streptavidin Boehringer,Mannheim, Ab Germany Magnetic particles −1 Polystyrene Bangs Labs,Fishers, IN, USA MPG 5 Porous glass —NH₂, Streptavidin, CPT, LincolnPark, NJ, USA hydrazide, avidin glyceryl Sera-Mag 1 Polystyrene —COOHStreptavidin Seradyn, Indianapolis, IN, USA SPHERO Various (1-Polystyrene —COOH, Secondary Abs Streptavidin, Spherotech, Libertyville,IL, magnetic particles 4.5) —NH₂ biotin USA XM200 3.5 Polystyrene —COOHSecondary Abs Protein A Advanced Biotechnologies, microsphere Epsom, UK

TABLE 2 End groups and Other Diameter Polymer activation Immobilizedimmobilized Manufacturer/ Name (nm) composition possibility antibodiescompounds supplier Ferrofluids 135, 175 Modified —COOH, SecondaryStreptavidin, Immunicon, hydrophilic —NH₂ Abs protein A HuntingdonValley, protein PA, USA MACS  50 Dextran —OH Secondary Streptavidin,Miltenyi Biotec, microbeads Abs, anti- biotin Bergisch Gladbach, CD AbsGermany Magnetic 90-600 Starch, —OH, Streptavidin, Micro-caps, Rostock,nanoparticles dextran, —COOH protein A, Germany chitosan biotin MagNIM 50, 250, —COOH, Secondary Streptavidin, Cardinal, Santa Fe, 500 —NH₂Abs, Ab protein A NM, USA against E. coli O157

In one embodiment of this method, both therapeutic cells and the lumensurface of the target vasculature are modified to include magneticallyresponsive particles. Either the therapeutic cells or the target surfaceis modified with permanently magnetized particles, and the compliment ismodified with permanently magnetized, ferromagnetic orsuper-paramagnetic or paramagnetic particles. Modifications may beaccomplished by cellular uptake of magnetic particles, by attachment orchemical conjugation of magnetic particles to the surface of therapeuticcells/target area surface as described herein for attachment molecules,or by attachment of vesicles/liposomes/micelles containing magneticparticles.

The magnetic particles can range from about 10 nm to about 10 μm indiameter. In one embodiment, the diameter of the particle is about 10 nmto about 1 μm, and in another embodiment about 50 nm to about 500 nm.The particles may comprise rare earth magnetic material, ferrouscomponents, and/or iron oxides.

The magnetic particles may be labeled with adhesion molecules, such asCD34, CD133, or antibodies thereof.

In one embodiment, a magnetically modified therapeutic cell is attractedinto the target area by magnetic force generated through an externalmagnetic field gradient. In a magnetic field with a gradient, magnetizedparticles, or magnetically responsive particles (such as paramagnetic orsuper-paramagnetic particles) are subject to a force in the direction ofthe gradient and proportional to the field gradient and the magneticmoment of the particle (in the case of paramagnetic particles, thismoment is induced by the external field, and thus the attractive forcealso becomes a function of the magnetic field strength as well as themagnetic susceptibility of the particle). If, for example, the targetvasculature is on the surface of the heart, and the target tissue is thelocal myocardium, then a magnetic field gradient directed perpendicularto the vascular wall and towards the target myocardium will exert aforce directed towards the vessel wall along the target tissue on themagnetically responsive particles. As described above, cells may bemagnetically modified by internalization of magnetic particles ofattachment of such particles to the surface of the cells. This may beaccomplished by using magnetic particles modified at their surface withantibodies to receptors present on the therapeutic (stem) cells. Suchparticles are commercially available.

Alternatively, magnetic particles may be introduced into the cell bymagnetic cationic liposomes (MCLs). Magnetic cationic liposomes areliposomes with a membrane with cationic lipids and are filled withmagnetically responsive nanoparticles. The electrostatic interactionbetween the cationic membrane and cell membranes results ininternalization of the nanoparticles into the cell. In addition,magnetically labeled cells may be imaged by MRI, thereby allowing toassess amount of cells retained in the target area and as well as theirspatial distribution.

E. Application of Magnetic Field Gradient Following Delivery ofTherapeutic Cells Comprising Magnetic Nanoparticles

A magnetic nanoparticle can be manipulated by an external magnetic fieldgradient (Pankhurst et al., J. Phys. D: Appl. Phys., 36: R167-R181(2003)).

In one example, the subject matter is directed to proactive retention ofcells by a surface chemistry that increases the mutual affinity. Theaffinity of the cell to the surface of the target vasculature isincreased or the system is modified to increase susceptibility tomagnetic attraction forces. Such changes result in an increased dwelltime, or residency, and increasing the stickiness. As such, thestickiness serves to retain or capture the therapeutic cells.

In one example, the therapeutic cells are combined with or infused withmagnetic particles using different methods. Ferrous oxide has beendemonstrated. The magnetic particles can be adhered to the therapeuticcells by various methods, including cellular uptake, attachment chemicalconjugation, incubation and attachment of vesicles.

A magnetic field applied externally can be used to direct the cells to atarget site. In various examples, either the endothelial cells, thetherapeutic cells or both the endothelial cells and therapeutic cellsare treated with the magnetic particles.

For example, with some mononuclear cells, magnetic particles of aparticular size are ingested. Ingestion can be facilitated withelectroporation, for example. Other commercially available tools, suchas magnetic beads, are affixed using surface adhesion. In one example,the therapeutic cells are modified with a specific antibody.

The magnetic attraction forces can be used to enhance engraftment. Forexample, the therapeutic cell mixture is mixed with magnetic beads andthen the beads attach specifically to the target cells and then thosecells are retained by a magnetic force.

In the present subject matter, the magnetic force is used to drawtherapeutic cells to a particular location on the target using amagnetic field gradient. For example, by establishing a magnetic fieldgradient directed perpendicular to the surface of the heart, followed byflushing the therapeutic cells, the cells will be directed to the vesselwall of the target vasculature.

The magnetic gradient is applied perpendicular using a static magneticfield source. In one example, the field is applied externally through anMRI-like magnet or a strong magnet external to the body. In one example,the magnetic field is oriented such that the field gradient is normal tothe surface of the heart in the target area. As such, the magneticparticles will experience a force normal to the surface of the heart.FIG. 3 illustrates organ 35 having damaged vessel 38. Static magneticfield gradient 30 is applied externally and exerts a magnetic force onmagnetic particles 39 in a direction substantially normal to vessel 38.In the figure, magnetic particles 39 are attached to therapeutic cells.

The magnetic beads used to enhance engraftment of therapeutic cells aremixed, and optionally labeled. In one example, the beads are labeled toattach them to the cells. Examples of labels include an antibody toCD-34, or CD-133, or an activated bead that has surface chemistry thatis amine-reactive.

The applied field generates a magnetic attraction that serves to slowthe flow in the localized region at the target and also tends toincrease the “adhesiveness” of a therapeutic cell to a target cell,which forces the therapeutic cells to dwell longer at the targetlocation. In one embodiment, a magnetic field may be applied for theduration of the infusion and possible dwell time. Infusion durationtimes may vary anywhere from 30 seconds to 1 hour. Exposure to magneticfields may be in similar time frame. For example, cells may be infusedin 30 second increments, followed by 3 minute occlusion periods. Afterthe therapeutic cells engage with the vessel wall, then the biologicalinteraction and the interaction between the receptor occurs.

F. Compositions, Dosages and Routes of Administration of TherapeuticCell Compositions

Compositions comprise therapeutic cells, including cells from differentsources, and optionally agents that enhance therapeutic cellengraftment, survival, proliferation and/or differentiation, enhancecardiac function or stimulate angiogenesis. The cells to be administeredmay be a population of individual cells or cells grown in culture so asto form a two dimensional or three dimensional structure. The number ofcells to be administered will be an amount which results in a beneficialeffect to the recipient. For example, from 10² to 10¹⁰, e.g., from 10³to 10⁹, 10⁴ to 10⁸, or 10⁵ to 10⁷, cells can be administered to, e.g.,injected, the target region of interest, for instance, infarcted andtissue surrounding infarcted tissue. Agents which may enhance cardiacfunction or stimulate angiogenesis include but are not limited topyruvate, catecholamine stimulating agents, fibroblast growth factor,e.g., basic fibroblast growth factor, acidic fibroblast growth factor,fibroblast growth factor-4 and fibroblast growth factor-5, epidermalgrowth factor, platelet-derived growth factor, vascular endothelialgrowth factor (e.g., VEGF₁₂₁, VEGF₁₄₅, VEGF₁₆₅, VEGF₁₈₉ or VEGF₂₀₆),tissue growth factors and the like. Such agents may optionally bepresent in the compositions or administered separately.

The cells are administered during a prophylactic, diagnostic ortherapeutic vascular procedure or an invasive or minimally invasivesurgical procedure. In one embodiment, the cells are administeredpost-myocardial infarction, within hours, e.g., 1 to 12 hours, to days,e.g., 1 to 2 days, and up to one or more weeks after myocardialinfarction. Preferably, the administration of therapeutic cells is priorto scar formation. The cells may be administered intravenously,transvenously, intramyocardially or by any other convenient route, anddelivered by a needle, catheter, e.g., a catheter which includes aninjection needle or infusion port, or other suitable device. Someexemplary delivery apparatus and methods include, but are not limitedto, the teachings provided herein.

In one embodiment, once administered, the therapeutic cells developfunctional connections with adjacent cells, membrane channels withadjacent cells, including viable cells in the recipient, and, if notalready differentiated, differentiate to myocardial cells.

Intracoronary catheter-based delivery of cells is known in the art. Forexample, the Transplantation of Progenitor Cells and RegenerationEnhancement in Acute Myocardial Infarction (TOP-CARE-AMI) pilot trialcompared the effect of direct intracoronary infusion of autologouscirculating progenitor cells and bone marrow cells in 20 patients whounderwent primary angioplasty for acute myocardial infarction. In thestudy, cells were delivered via an over-the-wire balloon catheteradvanced into a previously deployed stent.

III. Target Cells

To increase the probability and strength of attachment of therapeuticcells to cells located in the target site, i.e., target cells such asendothelial cells of the lumen surface of the target vasculature, thelumen surface may be modified such that the surface density of availableadhesion molecules is altered, e.g., increased. As discussed herein,target cell adhesion molecules possess an affinity to the surface of thetherapeutic cells, or molecular moieties thereof. Any of the methodologydisclosed herein for the modification of a therapeutic cell might beused to modify a target cell as well. For example, target cells may besubjected to mechanical conditioning, biological conditioning, chemicalconditioning, or any combination thereof. The expression of and/ornumber of endothelial adhesion molecules present on the surface of anendothelial cell such as ICAM-1, VLA-4 ligand (vascular cell adhesionmolecule-1 (VCAM-1)) and E-selectin can be altered by these methods.

Additional examples of adhesion molecules with an affinity to thesurface of the therapeutic cells include antibodies to adhesionmolecules present on the surface of therapeutic cells, e.g., anti-CD133or anti-CD34 antibodies.

In one embodiment, the expression profiles of adhesion molecules onendothelial cells is manipulated by the magnitude and type of shearstress, i.e., laminar v. turbulent, and time of exposure to the shearstress. Any mechanism that induces shear stress might be utilized in themechanical conditioning of target cells. For example, in one embodimentof mechanically conditioning target cells, a shear stress at rate of 20dynes/cm² increases the expression of ICAM-1, and decreases VCAM-1 andE-selectin expression on target endothelial cells through modulation oftranscriptional level gene expression (Chiu et al., Arterioscel ThrombVasc Biology, 24:1-8 (2004)). In addition, shear influences NF-kBtranscriptional factor in endothelial cells (Ganguli, A. et al., Circ.Res., 96 (6): 626-634 (2005)). In another embodiment, saline fluid canbe used to mechanically condition the target cells. In one example, thecatheter is loaded with saline, saline is infused into the vessel, andthrough a cyclic infusion/withdraw regime using a pump connected to thedistal end of the infusion catheter, shear is imposed onto endotheliumof the targeted tissue.

As for “biological conditioning,” target cells can be subjected toperiods of hypoxia to upregulate adhesion molecules. For example,intercellular adhesion molecule (ICAM)-1 and vascular cell adhesionmolecule (VCAM)-1 expression are upregulated in endothelial cellssubjected to hypoxia (Ng et al., Am. J. Physiol., 283: C93-C102 (2002)).In addition, hypoxia may directly activate NFk-B. NFk-B sites arepresent in the promoter of the ICAM-1 gene (Ohga et al., Nippon Risho,58:1587-1591 (2000)). Therefore, hypoxia may directly activate theICAM-1 through activation of NFk-B. Brief occlusions of targetvasculature may also result in the upregulation of adhesion moleculeexpression on endothelial cells. In addition, target cells at a targetsite in the vasculature may be genetically modified using any methodknown to the art to upregulate expression of an adhesion moleculesand/or to modify cell surface to enhance engraftment of therapeuticcells.

In addition, cells in the target area may be genetically modified priorto infusion of therapeutic cells to express (or increase the expressionof) adhesion molecules or to increase the surface presentation ofadhesion molecules. Genetic modification may be done by infusion ofviral vectors, liposomal or micelle delivery vehicles, plasmids, asdescribed herein. Intracoronary delivery of genetic material can resultin transduction of approximately 30% of the myocytes predominantly inthe distribution of the coronary artery. Parameters influencing thedelivery of vectors via intracoronary perfusion and enhancing theproportion of myocardium transduced include a high coronary flow rate,longer exposure time, vector concentration, and temperature. Genedelivery to a substantially greater percent of the myocardium may beenhanced by administering the gene in a low-calcium, high-serotoninmixture (Donahue et al., Nat. Med., 6:1395 (2000)). The potential use ofthis approach for gene therapy for heart failure may be increased by theuse of specific proteins that enhance myocardial uptake of vectors(e.g., cardiac troponin T). Improved methods of catheter-based genedelivery have been able to achieve almost complete transfection of themyocardium in vivo. Hajjar et al., (Proc. Natl. Acad. Sci. USA, 95:5251(1998)) used a technique combining surgical catheter insertion throughthe left ventricular apex and across the aortic valve with perfusion ofthe gene of interest during cross-clamping of the aorta and pulmonaryartery. This technique resulted in almost complete transduction of theheart and could serve as a protocol for the delivery of adjunctive genetherapy during open-heart surgery when the aorta can be cross-clamped.

Exemplary transgenes for delivery to a target cell include those genesthat express corresponding adhesion molecules to those adhesionmolecules found on a therapeutic cells, genes that express cytokines,genes that express Hypoxia Inducible Factor 1 (HIF-1), genes thatexpress heat shock protein (HSP) (for cellular protection), cytokines(SCF, HGF), chemokines (SDF1), chemokine receptors (CXCRs, CCRs),proteolytic enzymes (MMP-2, MMP-9), angiogenic growth factors (VEGFreceptors, such as VEGF-1/2/3/4; FGF receptors, such as FGFR-1/2/3/4),and anti-apoptosis (akt).

In one embodiment, a “two-step procedure” can be used to biologicallymodify a therapeutic cell and a target cell. For instance, cells at thetarget site can be modified, e.g. by delivery of a gene that wouldenhance expression of a counter-receptor; which modification is followedby the delivery of therapeutic cells. For example, on day 1, a gene ofinterest is delivered to a target site, and on day 2, therapeutic cellsare infused. Thus, sufficient time for expression of the delivered geneis allowed.

In another embodiment, bi- or multifunctional linker molecules areinfused/injected into the target area vasculature prior to, orsimultaneous with, the infusion of therapeutic cells, where at least onefunctionality of the linker molecule has affinity to the surface of thelumen surface of the target area vasculature, e.g., endothelial cellsurface, and at least one other functionality has affinity to thesurface of the therapeutic cells, as illustrated in FIGS. 4A and 4B.FIG. 4A illustrates endothelial cells having a surface modified withbi-functional linker molecules. FIG. 4B illustrates therapeutic cellsattached to a lumen surface by bi-functional linker molecules. Toenhance accessibility, the functionalities may be separated by a spacer,such as a hydrophilic polymer chain, e.g., PEG. For multifunctionallinkers, the spacer may have branches or be of star form.

For example, a CD133-antibody linked via a PEG spacer to a CD31-antibodyis used to modify the surface of an endothelial cell resident at thelumen surface of the target vasculature. The CD31 antibody has affinityto the CD31 receptor present at the surface of the endothelial cells,while the CD133 antibody has affinity to the CD133 molecule on thesurface of the therapeutic cell, e.g., a therapeutic stem cell. Tomodify the surface of the endothelial cell, the bifunctionalantiCD133-PEG-antiCD31 compound is infused into the target vasculature.As the antiCD31 moiety attaches to the CD31 receptor, the endothelialcell surface will effectively present anti-CD133 antibodies withaffinity to the surface of an endothelial cell found in the targetvasculature.

Alternatively, a homo-bifunctional linker molecule is used. For example,two anti-CD34 antibodies are joined by a short carbon linker and areused to modify the surface of resident endothelial cells ofmicrovasculature. While one of the two antibody moieties adheres to CD34present on the surface of capillary endothelial cells, the otheranti-CD34 moiety is presented outward. Therapeutic cells having CD34 ontheir surface bind to endothelium pretreated in this fashion.

In another embodiment, molecules or molecular moieties possessingaffinity to the surface of the therapeutic cells may be chemicallyconjugated to the luminal surface of the target area vasculature, asillustrated in FIGS. 5A and 5B. FIG. 5A illustrates surface modificationof lumen cells using NHS reactive linker molecules. In the figure, theNHS linker molecules each include NHS and anti-T. FIG. 5B illustrates atherapeutic cell attached to an endothelial cell of the targetvasculature. A molecule or molecular moiety is conjugated to the lumensurface via a spacer molecule to enhance accessibility. A molecule maypossess more than one molecular moiety with affinity to the targetsurface, in which case the spacer may be branched. Chemical conjugationis achieved by infusion of said molecules into the target areavasculature. To enhance the efficiency of the conjugation, the targetarea vasculature is flushed with saline prior to infusion of attachmentmolecules. Attachment molecules may be chemically conjugated, i) toamine groups using reactive esters, epoxide, ii) to sulfhydryl groupsusing maleimides, vinyl sulfones, or iii) to carboxyl groups usingdimethylaminopropyl-carbodiimide (EDC) chemistry.

For example, the lumen surface of the target vasculature (e.g. capillaryendothelial cells) may be modified by antibodies to receptors present onthe surface of the therapeutic, e.g., stem cells (e.g. CD34, CD133,KDR). This may be done by infusing a VS-PEG-antibody molecule into thetarget vasculature and thereby, conjugating the antibody to sulfhydrilgroups present on the surface of endothelial cells of the targetvasculature. A VS-PEG-antibody molecular construct may be made asdescribed above.

In additional embodiments, other antibodies, fragments thereof, ormolecules other than antibodies may be conjugated to the lumen surfaceof the target vasculature.

Molecules or molecular moieties possessing affinity to the surface ofthe therapeutic cells may be introduced and anchored in the membrane ofendothelial cells of the target area vasculature by liposomal or micelledelivery.

For example, CD31 antibodies or fragments thereof may be conjugated to aphosphatidyl ethanolamine lipid with di-C16 or longer chains. Theselipid-antibody conjugated may be embedded in micelles or liposomes andinfused into the target area. Alternatively, hydrophobic peptidealpha-helices (such as poly-leucine) may serve as membrane anchors.

Circulating endothelial progenitor cells amass at sites of injury(Asahara et al., Science, 275:964-966 (1997); Asahara et al.,Circulation Research, 85: 221-228 (1999)). Sites of injury arecharacterized by local cell irritation. The infusion of mild irritants,such as slightly acidic or basic buffers, diluted ethanol, and lacticacid, prior to infusion of the therapeutic cells to the target area maytherefore increase cell retention. In one embodiment, an agent such asbut not limited to ethanol (diluted to 0.01-0.5% by volume); an acidicbuffer (i.e., a buffer having a pH in the range of about 5.5 to about7.0, e.g., pH 6.5±0.5; a basic buffer (i.e. a buffer having a pH in therange of about pH 8.0 to about pH 9.0, e.g., pH 8.0-8.5; highconcentration saline (i.e., a saline solution in the range of about 180mM to about 300 mM NaCl, e.g., about 200-250 mM NaCl); a heatedsolution, e.g. a saline solution in the range of about 38-42° C., e.g.,about 39-40° C.

In another embodiment, the infusion of stimulants, such as cytokines,chemokines, growth factors, hormones, nitric oxide (NO) and othermessenger molecules prior to infusion of the therapeutic cells to thetarget area may increase cell retention.

IV. Induction of Transient, Localized Ischemia

In certain embodiments, the subject matter includes devices and methodsthat provides a means of delivering therapeutic cells, e.g., previouslyprepared cells, to a target site, such as the heart, and promoting theengraftment, e.g., absorption, of the cells into the target area, e.g.,into the myocardium. The cells may be intended to produce any number ofdifferent effects. One example is to promote myocardial regenerationfollowing an infarct by causing therapeutic cells to be absorbed by theheart.

Current methods focus on initially establishing ischemic conditions inorder to activate receptors that promote call absorption. This isaccomplished by inflating an occlusion balloon within the coronaryvasculature. The balloon is later deflated and cells are then injectedinto the coronary arteries. The cells travel down stream to the ischemicregion to be absorbed. This method is undesirable in several respects.First, the necessary vessel occlusion introduces a level of patient riskwhile at the same time making the patient extremely uncomfortable.Second, once the therapeutic cells are introduced, they readily flowpast the ischemic region such that the opportunity for absorption isminimal. The concepts presented herein are intended to address theseconcerns. Each provides a “controlled” method of introducing ischemiawhile increasing the “soak time” such that the opportunity for cellabsorption is also increased.

Herein a device is described to inject medical grade carbon dioxide(CO₂) into the desired artery. CO₂ is an established alternateangiographic contrast agent, and can be delivered by pump or injection(see Cronin et al., Clin. Radiol., 60: 123-125 (2005)). Rather thandelivering a bolus of CO₂, the device described herein delivers the gascontinuously in the form of a stream of small bubbles. The CO₂ bubblesinduce a localized, hypoxic environment in the arterial and/or venialsystem. By “small bubbles” or “microbubbles” is meant a bubble that canpass through a capillary, for example, a CO₂ bubble having a diameter ofless than about 6 microns (typically about 3 microns—about 6 microns).In one embodiment, CO₂ microbubbles of the size of ultrasound contrastmedia can be used. In addition, microbubbles larger than 6 microns canbe used to occlude microvessels temporarily until the microbubbledisintegrates and blood flow resumes, which occlusion will create anischemic environment due to the occlusion of flow and due to elevatedcarbon dioxide generated by the bubble. The lack of flow will alsofacilitate therapeutic cell adhesion in the vicinity of the ischemicenvironment.

If necessary, these bubbles may be small enough and plentiful enough tocreate a dispersion between the flowing blood and the CO₂. The CO₂bubbles displace the blood such that ischemia is introduced, however,the artery remains patent such that blood continues to flow.

The bubble infusion rate is adjusted as a means of regulating the levelof ischemia (e.g., by active regulation of by use of a predetermined,fixed setting). A steady-state level of ischemia is established thatproduces the necessary preconditioning (receptor activation) at thetarget site. The time required to absorb the CO₂ will result in anoverall reduced flow rate of the blood/CO₂ combination. Thus a reducedlevel of perfusion is also established.

In one embodiment, previously prepared therapeutic cells (stem cells,for example) are injected alongside the CO₂ via the same catheter suchthat the blood/CO₂ mixture now becomes a mixture of blood, CO₂, andcells. Because some reduced level of perfusion remains present, thecells are slowly carried down stream and enter the ischemic capillaryregion. They slowly pass across this region and are absorbed byreceptors activated by the ischemic condition.

The CO₂ component maintains the required ischemic level to promote cellabsorption. The reduced flow rate provides additional time for the stemcells to be absorbed as they pass across the ischemic region.

Thus, in one embodiment a method entails a continuous process ratherthan a repetitive series of vessel occlusions followed by stem cellinjections. In addition, a myocardial infarct site can be readilytargeted by advancing the device more distally than a device thatincludes the use of a balloon.

In an alternative embodiment, the “dispersion” is created by passing theCO₂ through a small sponge contained within the distal tip of thecatheter, as illustrated in FIG. 6B.

In one embodiment, saline is used to introduce ischemia.

In one example, therapeutic cells and CO₂ microbubbles are delivered toa target site via a catheter. The CO₂ microbubbles burst and deliver CO₂to a localized target area, which preconditions the target site toenhance the engraftment of the therapeutic cells.

In one example, the CO₂ microbubbles are delivered in a continuousstream by using a porous element of a catheter head. The porous element,in one example, includes a sponge-type material or a porous ceramic orsynthetic material.

FIG. 6A illustrates exemplary catheter system 60 having implantablemixing chamber 64 at a distal end of catheter body 62 and dispersionport 66 and therapeutic cell port 68 at a proximate end. Catheter body62, in the example illustrated includes two lumens. Dispersion port 66is configured to receive gaseous CO₂ and therapeutic cell port 68 isconfigured to receive therapeutic cells. Port 66 and port 68, in variousexamples, are coupled to a pump or syringe.

FIG. 6B illustrates bubble producing mixing chamber 64 receives adispersion via line 72 and therapeutic cells via line 74A. Line 72 iscoupled to dispersion port 66 and terminates within reservoir 80 atbubbler 76. Bubbler 76, in the example illustrated, includes a spongematerial, however other bubble producing materials can also be used.Line 74A is coupled to therapeutic cell port 68 and terminates withinreservoir 80 at end 74B. End 74B, in the figure, is a plain end andreleases stem cells denoted herein by the symbol “s.” Blood entersreservoir 80 at entry port 78. Blood entering reservoir 80 mixes withthe bubbles formed by bubbler 76 and therapeutic cell(s) from end 74Band exits mixing chamber 64 at discharge port 82 in the form of adispersion.

In one example, the CO₂ bubbles occlude the capillary flow.

In one example, ischemia is induced using a catheter to inject CO₂ in astream of tiny bubbles. In one example, medical grade CO₂ is injectedinto an artery in a stream of tiny bubbles having a foam-likeconsistency. The bubbles in the foam are sufficiently small toapproximate an dispersion of blood and CO₂. The concentration of CO₂required to induce ischemia can be calculated based on the concentrationof oxygen required in the region. A pump or valve can be used to controlthe perfusion, or flow rate, of solution into the vasculature.

According to one theory, the time required to absorb the CO₂ willeffectively reduce the flow rate of the blood and CO₂ combination. Assuch, a reduced level of profusion is established. Reduced level ofprofusion is established since the CO₂ displaces the blood flow and thetherapeutic cells that are being introduced.

According to one theory, CO₂ micro bubbles and therapeutic cellsincrease Brownian motion, thus increasing the opportunity for cells tobump against the surface of the vessel wall. In one example, the CO₂micro bubbles and therapeutic cells forms an dispersion that isdelivered in a single continuous process.

In one example, the target endothelium is preconditioned by introducingthe CO₂ followed by a bolus of therapeutic cells. The flow rate can bemodulated to achieve a desired engraftment. In one example, the rate ofblood flow is controlled and maintained at a non-zero level to induce aregional ischemic event.

A. Microsphere Induced Occlusion

In one embodiment, a device is placed proximal to the target infusiontissue. A controlled release of microsphere materials are released intothe vessel. These spheres are delivered to the capillary bed and occludeor “plug up” the capillary bed. By plugging up or occluding some of thecapillaries, the amount of oxygen delivered is reduced and ischemia isthereby created. Thus, an appropriate delay is elapsed, such as the timein which blood in the target tissue is displaced, to induce controlledischemic conditions. In one embodiment, microspheres of 9-15 microndiameter may be used to occlude capillaries. The microspheres occlusionmay be such that all flow is blocked in the capillary. In this case,lesser amounts of oxygen may be delivered by adjacent unoccludedcapillaries, thereby creating an ischemic environment. In anotherembodiment, the microspheres may block the capillary in such a way thatred blood cells cannot pass through and deliver large amounts of oxygen,but blood plasma can pass through to deliver very small amounts ofoxygen, again creating an ischemic environment. In one embodiment,microspheres may reduce oxygen delivery by 50 to 75%. In anotherembodiment, the microspheres may reduce oxygen delivery by 60-95%. Inanother embodiment, the microspheres may be composed of a biodegradableor bioabsorbable material that would limit the duration of the occlusionto a few days. Therapeutic cells are then introduced into the targettissue and allowed to “soak” for an appropriate period of time. Thespheres are then deactivated, and normal blood flow resumes to thetissue.

In one embodiment, this process is controlled by a blocking balloon onthe delivery device.

In another embodiment, this process is computer controlled withautomatic timing, injections, and monitoring of EKG for proper anddangerous ischemic conditions.

In one embodiment, the microspheres are dissolvable and in one example,are made of bioabsorbable material that is absorbed at different periodsof time. Examples of bio-absorbable materials include, but is notlimited to, degradable polymers such as polycaprolactone, PLGApoly(lactide-co-glycolide), Polyester-amide, polyphosphazine, tyrosinecarbonate, etc., or Alginate crosslinked with divalent Ca, Ba or Srcations. The microspheres may also be made of an extra-cellular matrixprotein such as collagen or gelatin, crosslinked with glutaraldehyde toprevent quick dissolution.

The microspheres occlude the target site and are later dissolved uponapplication of energy or after a period of time. For example, byapplying thermal energy, a solvent, ultrasonic energy or radio frequencyenergy, the microspheres dissolve. In one example, the microspheresdissolve upon exposure to a particular temperature.

In one example, the fluid flow is temporarily occluded after infusion oftherapeutic cells. In one example, shear forces are exerted on both theendothelial cells and the therapeutic cells based on the relativemovement there between. Accordingly, as the flow rate increases, theshearing forces increase. To reduce the flow rate, a flow resistor isplaced in the lumen of the vasculature. The flow resistor is positionedupstream relative to the target site, however, in one example, the flowresistor is positioned downstream.

In one example, the flow rate is controlled by retro-profuse cells atthe target site. Retro-profusion entails a distal occlusion whichreverses the fluid flow. The reversed fluid flow occurs as a result ofpressure applied to drive the cells up the vascular bed rather thandownstream.

In various examples, therapeutic cells are delivered through a catheter.The target vessel is occluded for a brief time period (three minutes inone example) to induce ischemia at the target site. Following occlusion,therapeutic cells are delivered.

In one example, the fluid flow is temporarily stopped and thenimmediately thereafter therapeutic cells are infused. In one example,the vessel is occluded after the time of infusion such that an effectivenumber of the infused therapeutic cells are located at the target sitewhen the flow is at a reduced rate. The target area is a short distancefrom the infusion site. In one example, the occlusion is establishedbetween approximately 0.5 and 2 seconds after infusion. Times greater orless than 0.5 and 2 seconds are also contemplated. In one example, amajority of therapeutic cells are at the target site when the occlusionoccurs.

The therapeutic cells will be spatially distributed in the vessel soonafter the time of infusion. The delay between therapeutic cell infusionand vessel occlusion is selected to provide that the bulk of therapeuticcells are in the target region at the time of vessel occlusion.

In one example, therapeutic cells are infused in a sequence of pulsesduring which the flow rate in the vessel is varied between fullyoccluded and no resistance. In such an example, the therapeutic cellsarrive in multiple groups and the occlusion occurs in a time sequencesuch that each group briefly pauses for a period of time as it travelsthrough the target area. In one example, an estimate of the flow rateinforms the decision as to when the therapeutic cells are injected as afunction of the location of the infusion site relative to the targetsite and when the vessel is occluded. A typical flow rate for a healthyheart is approximately 40 ml per minute which will vary with vessel sizeand location. In one example, the flow rate is measured using a sensoror imaging of the vasculature.

Various catheter designs can be used to introduce an ischemia producingagent, such as CO₂. FIGS. 6A and 6B illustrate one such example tailoredto generate small CO₂ bubbles by forcing pressurized CO₂ across amembrane. The membrane is selected to have a micro porosity to formextremely small bubbles that float into capillary beds and momentarilyplug the capillary beds to cause ischemia. In one method, an arteryflowing into the target area for delivery of therapeutic cells ismomentarily plugged for some predetermined amount of time using CO₂,which causes more ischemia, which is then followed with therapeutic celldelivery.

In various example, the capillary bed is plugged or occluded eitherpartially or wholly to fluid flow. The capillary bed can be occludedusing a flow resistor as described elsewhere in this document.

According to one theory, the CO₂ dispersion flowing through thevasculature is absorbed in a gas exchange in the lungs due to vaporpressures.

In one example, the level of ischemia in a target location is monitoredusing a sensor. The sensor output is used in a feedback loop to controlthe level of ischemia. The level of ischemia can be regulated byadjusting the resistance of an element or by changing a concentration ofCO₂ or applied pressure. In one example, the feedback signal isgenerated using an oxygen sensor positioned on the back side of thecapillary bed in a vein.

According to one theory, introduction of a CO₂ dispersion is effectiveto create increased Brownian motion. Brownian motion refers to therandom movement of microscopic particles suspended in liquids or gassesresulting from the impact of molecules of the fluid surrounding theparticle. Brownian motion causes increased “bumping,” which refers tothe manner in which the therapeutic cells travel along the endothelialtarget area, thus increasing the likelihood of bonding with an adhesionmolecule.

A catheter having a sponge and a mixing chamber is illustrated in FIGS.6A and 6B. The catheter includes multiple lumens and in the exampleillustrated, one lumen is used to inject CO₂ and the other lumen is usedto inject the therapeutic cells into the catheter. The first lumenterminates in a sponge that serves to generate micro bubbles. The mixingchamber receives the therapeutic cells, blood and the CO₂, thus forminga dispersion.

Other examples are also contemplated, including, for example, a catheterhaving multiple lumens in which the CO₂, the therapeutic cells and bloodare combined. The CO₂, in various examples, would be in the form ofmicro bubbles. A vent is provided to allow blood to fill into the mixingreservoir. Other methods and structures are also contemplated forenhancing mixing in the chamber before delivery to the vasculature.

According to one example, the CO₂ is injected and mixed with blood andtravels downstream to the target area. The CO₂ causes ischemia of thevessel. In one example, the CO₂ is introduced in a series of smallinjections interspersed by delay periods.

Other delivery regimens are also contemplated. For example, after aperiod of time during which the CO₂ is absorbed and the blood flowresumes, another bolus of CO₂ is introduced along with therapeuticcells. The dwell time of the therapeutic cells can be controlled by theCO₂ delivery regimen. In one example, a thickener, gel or other agent isadded in the mixing chamber of the catheter to increase the viscosity.An exemplary thickener includes CO₂ foam, micro spheres or liposomes.

In one example, micro spheres include small spheres of a flow occludingsubstance that can later be dissolved or dispersed. For example, microspheres fabricated of albumin (e.g., Optison) will disperse or dissolvewhen subjected to an externally applied field of ultrasonic energy orradio frequency energy. As another example, micro spheres fabricated ofpoly(lactide-co-glycolide) (PLGA) will biodissolve after a period oftime.

According to one theory, the micro spheres occlude or plug the capillarybed since their dimensions are physically too large to pass through thebed.

To form the CO₂ bubbles, CO₂ is forced over a porous membrane or thesponge. FIG. 6B illustrates gas bubbler 76 fabricated of a spongematerial disposed within a reservoir of a mixing chamber. Otherconfigurations are also contemplated, including a dual reservoir mixingchamber. In such an example, one reservoir receives CO₂ and the otherreservoir receives the therapeutic cells. The CO₂ reservoir includes aporous medium through which bubbles are formed. One lumen is ported tothe chamber. Another port allows blood from the artery to bypass or flowinto the chamber.

In operation, the catheter perfuses CO₂, blood and therapeutic cells. Inone example, a lumen of a catheter is terminated with a porous mediumthrough which stem cells, blood and carbon dioxide bubbles perfuse. Inone example, blood enters through a bypass port and therapeutic cellsare injected into a chamber concurrent with CO₂ bubbling and the mixtureis discharged from the chamber at the distal end of the catheter. Inaddition to blood, CO₂ and therapeutic cells, other materials can bedelivered using the catheter of the present subject matter. For example,a drug or other agent to cause ischemia or otherwise improve engraftmentcan be delivered using the present catheter either with or without theporous medium. The drug or other agent can be a gaseous, solid or liquidsubstance.

V. Alternative Examples

In addition to the examples presented above, other embodiments are alsocontemplated.

In an alternative embodiment, a “therapeutic cell” includes atherapeutic drug carrier such as a liposome or a polymer particle with asurface molecule that has an affinity to the lumen surface of thevasculature.

In one example, the present subject matter entails surface recognitionthrough surface tailoring. For example, surface tailoring of the boththe therapeutic cell and the target cell is employed to enhanceengraftment. The surface modification methods presented elsewhere inthis document can be combined synergistically such that, for example,the therapeutic cell surfaces are modified to present molecular moiety A(for example, through genetic modification, surface modification orother methods) and the lumen surface of the target vasculature area(endothelial cells) are modified to present molecular moiety B wheremoiety A has affinity to moiety B. Moiety A does not necessarily have anaffinity to endothelial surface and moiety B does not necessarily havean affinity to the surface of the therapeutic cells). For one example,avidin is conjugated to one surface and biotin is conjugated to thecomplementary surface. Biotin may be conjugated to lysines of membraneproteins present at the cellular surface using NHS-PEG-biotin molecules.Avidin may be bound to the biotin present on the cell surface using anadditional incubation in avidin, effectively immobilizing avidin at thecell's surface.

All patents and publications referenced or mentioned herein areindicative of the levels of skill of those skilled in the art to whichthe subject matter pertains, and each such referenced patent orpublication is hereby incorporated by reference to the same extent as ifit had been incorporated by reference in its entirety individually orset forth herein in its entirety. Applicants reserve the right tophysically incorporate into this specification any and all materials andinformation from any such cited patents or publications.

What is claimed is:
 1. A method of enhancing engraftment of therapeuticcells at a target site in the lumen of the vasculature of a mammal,comprising: contacting the therapeutic cells with a biological linkerhaving at least two functionalities, wherein the linker is attached tothe cell membrane of the therapeutic cells through the affinity of atleast one functionality of the linker to the surface of the therapeuticcells, wherein at least one other functionality of the linker hasaffinity to the lumen surface of the target area vasculature, whereinthe at least two of the functionalities of the linker are separated by aspacer having branches or being of star form and placing the linkerattached to the therapeutic cells at the target site.
 2. The method ofclaim 1, wherein the linker is irreversibly attached to the therapeuticcells.
 3. The method of claim 1, wherein the linker is reversiblyattached to the therapeutic cells.
 4. The method of claim 1, wherein thelinker is a biological conjugate.
 5. The method of claim 1, wherein thelinker comprises more than two functionalities.
 6. The method of claim1, wherein the linker is a bi-functional linker.
 7. The method of claim1, wherein the spacer is a hydrophilic polymer.
 8. The method of claim1, wherein the spacer comprises PEG.
 9. The method of claim 1, whereinthe linker comprises an affinity molecule selected from the groupconsisting of linked antibodies, F_(ab) fragments of antibodies,affibodies, peptides, and combinations thereof.
 10. The method of claim9, wherein the affinity molecule has an affinity to a cell-surfacemolecule on a cell at the target site in the lumen, wherein thecell-surface molecule is selected from the group consisting of PECAM(CD31), vascular cell adhesion molecule-1 (VCAM-1), intercellularadhesion molecule (ICAM-1), a selectin, P-Selectin (CD62P), E-Selectin(CD62E), L-selectin, Flk-1 and combinations thereof.